Seeds of Arabidopsis thaliana Columbia-0 (WT), myb30-1 (SALK_061315), and myb30-2 (SALK_027644C) (a T-DNA insertion in the 5′-UTR of At3g28910 in the Col-0 background) were sourced from the Biological Resource Center of Arabidopsis. Pro35S:MYB30-Flag/Col-0 transgenic lines were constructed by transforming the Pro35S:MYB30-Flag vector into Col-0 via Agrobacterium strain EHA105-mediated infiltration. Two independent transgenic lines in T2 progeny, MYB30-OX 1 and MYB30-OX 2, were used in the present research. The related primers are shown in Supplementary Table 1.

The seeds of the plants were sterilized on the surface using 70% ethanol, rinsed with sterile distilled water, and then planted on Murashige and Skoog (MS) culture with 2.5% sucrose and 0.3% phytagel agar (Sigma-Aldrich, St. Louis, USA). Plates were kept at 4 °C for 2 days and then transferred to constant illumination at 23°C. For phenotypic assays, 4-day-old plants were vertically grown on MS culture (pH = 5.8) with or without iron (36.7 mg/L of FeNaEDTA). MS culture (Cat PM1011) and MS-Fe (Cat PM1061-Fe) culture were purchased from Coolaber Science & Technology (Beijing, China). Photographs were taken and root length was scored after 5 days of growth using ImageJ (https://imagej.nih.gov/ij/).

Chlorophyll content was measured using previously described methods (Arnon, 1949). Briefly, plant shoots were ground to a powder in liquid nitrogen and then resuspended in 80% (v/v) acetone and centrifuged at 10,000g at 4 °C for 10 min. The spectroscopy absorbance was measured at 646 and 663 nm.

For Fe concentration determination, plant roots and leaves were dried at 70 °C for 3 days. Approximately 0.1 g of dried tissue was digested with a SpeedWave Two (Berghof Products, Eningen, Germany) using a mixture of hydrogen peroxide and HNO₃ (4:1, v:v) at 200 °C for 60 min. Fe concentration was measured by atomic emission spectroscopy.

Malondialdehyde (MDA) was determined using a plant MDA assay kit (Solarbio, Beijing, China, Cat BC0025). Following the kit’s instructions, approximately 0.5 g of the sample was weighed, and 0.5 mL of the extraction solution was added for homogenization in an ice bath. The mixture was then centrifuged at 8,000g for 10 min at 4°C. One hundred microliters of the supernatant was taken, and 300 μL of MDA detection working solution and 100 μL of reagent 3 were added, mixed well, incubated in a 100°C water bath for 60 min, cooled to room temperature, and centrifuged at 10,000g for 10 min. The absorbance of each sample was measured at 450, 532, and 600 nm, and the content of MDA was calculated. In the control tube, 100 μL of distilled water was added for the reaction.

Reactive oxygen species (ROS) were measured in terms of H₂O₂ levels. For H₂O₂ histochemical detection, seedlings were incubated overnight in the dark with 1 mg/mL of 3,3′-diaminobenzidine (DAB) solution (pH = 3.8). Seedlings were immersed in 95% ethanol for 12 h to remove chlorophyll.

H₂O₂ levels were assessed using a Solarbio (Beijing, China) H₂O₂ Content Assay Kit (Cat<ns/> BC3595). Approximately 50 mg of fresh tissue sample was powdered with 500 μL of cold acetone. Following a 10-min centrifugation at 8,000g at 4 °C, the supernatant was extracted for examination. A 250-μL aliquot of the supernatant was taken from the test tube, and 25 μL of reagent 2 and 50 μL of reagent 3 were added, after which the mixture was reacted in a room-temperature water bath for 10 min. The sample was then centrifuged at 4,000g for 10 min, and the supernatant was discarded. Subsequently, 250 μL of reagent 4 was added to dissolve the precipitate, and the absorbance value was measured at a wavelength of 415 nm to calculate the H₂O₂ content. For the control tube, 250 μL of reagent 1 was added.

Enzymatic antioxidant activity was determined in 4-day-old seedlings with or without iron deficiency treatment for further growth for an additional 5 days. The activities of peroxidase (POD, Cat A084-3-1), superoxide dismutase (SOD, Cat A001-4), and catalase (CAT, Cat A007-1-1) were determined using assay kits from Jiancheng (Nanjing, China) following the kit’s instructions.

For POD detection, approximately 0.1 g of fresh tissue was taken, and 9 times the volume of physiological saline was added according to a weight (g) to volume (mL) ratio of 1:9. Then, 10% tissue homogenate was prepared under an ice-water bath and centrifuged at 3,000 rpm for 10 min. Next, 0.1 mL of the supernatant was taken, and 2.4 mL of reagent 1, 0.3 mL of reagent 2, and 0.2 mL of reagent 3 were added. After reaction at 37 °C for 30 min, 1 mL of reagent 4 was added to terminate the reaction. The supernatant was then taken, and the absorbance was measured at 420 nm to calculate the enzyme activity. In the control tube, reagent 3 was omitted. For CAT detection, after the tissue was homogenized, 0.1 mL of the supernatant was taken, and 1 mL of pre-warmed reagent 1 at 37 °C and 0.1 mL of reagent 2 were added. After reacting for 1 min, 1 mL of reagent 3 and 0.1 mL of reagent 4 were added and mixed well, and the absorbance at 405 nm was measured to calculate the enzyme activity. The control tube was finally added with the tissue homogenate. For SOD detection, 0.2 g of fresh tissue was taken, and 4 times the volume of homogenization medium (reagent 8) was added. A 20% tissue homogenate was prepared under an ice-water bath and centrifuged at 3,500 rpm for 10 min. A 0.1-mL aliquot of the supernatant was taken and diluted 3 times with reagent 8. Then, 60 μL of the diluted solution was taken, and 1 mL of reagent 1, 0.1 mL of reagent 2, 0.1 mL of reagent 3, and 0.1 mL of reagent 4 were added, mixed well, and reacted at 37 °C for 40 min. Two milliliters of chromogenic reagent was added, and the absorbance was measured at 550 nm to calculate the enzyme activity. The control tube was added with the corresponding volume of reagent 8 for the reaction.

Plant metabolites were extracted from the roots of 4-day-old WT and myb30–2 seedlings with or without iron deficiency stress for an additional 5 days for further growth, quantified by GC-MS time-of-flight (TOF) and analyzed as described previously (Watanabe et al., 2013), using the RBioconductor package gpfortify to construct heat maps and principal component analysis (PCA). Metabolites that showed significant changes were defined as previously described (Benjamini and Hochberg, 1995). Metabolomics pathway analysis (MPA) was calculated from relative concentrations after removing non-changing data between WT and myb30–2 lines under iron-deficient conditions.

Total RNA was isolated from treated seedlings using TRIzol reagent from TianGen (Beijing, China). One microgram of this RNA was employed for reverse transcription with M-MLV reverse transcriptase (Vazyme, Nanjing, China). The gene expression levels were determined using the SYBR quantitative PCR Mix on a Roche quantitative instrument (Rotkreuz, Switzerland). Relative mRNA levels were assessed using the 2^–ΔΔCt method, with ACTIN2 serving as the internal reference. The primers are listed in Supplementary Table 1.

Seedling proteins were extracted in RIPA lysate buffer (Biosharp, Hefei, China), separated via SDS-PAGE, and transferred to a PVDF membrane at 80 V for 2 h. Blots were treated with Flag antibody (1:4,000, Cell Signaling Technology, Danvers, USA) overnight at 8°C and then incubated with the secondary antibody (horseradish peroxidase-conjugated) for 1 h at 25°C, using a chemiluminescence reagent to visualize the proteins.

One-way ANOVA test was used to analyze the statistical significance, with lowercase letters indicating significant differences (P < 0.05). The results are displayed as means ± SD. Root length measurements were assessed through nine seedlings per group (n = 9). For measuring chlorophyll content, Fe concentration, MDA content, and ROS content, approximately 0.5 g of shoots per group (n = 3) were used.

To molecularly verify the involvement of MYB30 in the plant response to iron deficiency stress, 4-day-old seedlings of Col-0 were transferred to iron-deficient MS medium for further growth observation. Using fluorescence qRT-PCR, we found that the transcription level of the MYB30 gene was elevated under iron-deficient conditions and increased progressively with treatment time. After 3 days of iron deficiency treatment, MYB30 expression increased approximately sevenfold. Consistent with this, MYB30 protein abundance also increased significantly with prolonged iron deficiency treatment (Figure 1). These results indicate that MYB30 is activated at both the transcriptional and translation levels under iron deficiency, suggesting its potential role in the plant’s response to this stress.

To investigate the regulatory role of MYB30 under iron-deficient conditions, we examined the phenotypes of Col-0 and MYB30 loss-of-function mutants (myb30–1 and myb30-2; Supplementary Figure 1A). When grown on standard MS medium, both mutants displayed growth similar to Col-0. Under iron-deficient conditions, however, all plants developed smaller leaves and shorter primary roots (Figure 2). Notably, the length of the primary roots of myb30–1 and myb30-2 was significantly shorter than that of Col-0, indicating enhanced sensitivity to iron deficiency.

To further confirm the role of MYB30, we used MYB30 overexpression lines (MYB30-OX, 1 and 2; Supplementary Figure 1B). Under sufficient iron, these lines grew similarly to the control. Under iron deficiency, however, their primary roots were significantly longer than those of Col-0 (Figure 2). Collectively, these results showed that MYB30 positively influences the plant response to iron deficiency stress, primarily by promoting root growth to enhance adaptation to low-iron environments.

Iron deficiency leads to incomplete chloroplast structure, decreases photosynthetic capacity, and eventually causes huge losses in crop quality and yield (Ladygin, 2004). To assess whether MYB30 influences chlorophyll accumulation under iron deficiency, we measured chlorophyll content in plant shoots. All seedlings showed reduced chlorophyll content under iron deficiency, but the decrease was most pronounced in myb30 plants. In contrast, MYB30-OX plants maintained higher chlorophyll levels (Figure 3). This suggests that MYB30 improves chlorophyll accumulation under iron deficiency.

Although essential, iron can also be toxic due to its reactivity (Morrissey and Guerinot, 2009). ROS production is directly related to lipid peroxidation (Ma et al., 2015), which can be assessed by detecting MDA levels (Blokhina et al., 2003). Under iron deficiency, MDA content in the roots and shoots was significantly higher in myb30 plants compared to the control, while MYB30-OX plants displayed a reverse trend (Figure 4). This indicates greater oxidative damage in myb30 plants under iron-deficient conditions.

Together, these findings show that myb30 plants exhibit reduced root elongation, lower chlorophyll content, and increased lipid peroxidation under iron deficiency. To uncover the mechanisms behind these responses, further metabolomics profiles were analyzed.

Using GC-MS metabolite profiling, we evaluated metabolomic changes between the Col-0 and myb30 plants under iron deficiency. Among the 840 metabolites present in all samples, 134 were identified (Supplementary Table 2). To analyze the metabolic differences between Col-0 and myb30 plants, PCA was used to find that the first component accounted for 82.3% of total variance (Figures 5A, B). Hierarchical clustering of relative metabolite concentrations further demonstrated distinct abundance patterns between Col-0 and myb30 plants under iron deficiency (Figure 5C).

Furthermore, we conducted a comprehensive analysis of pathways altered in myb30 plants under iron deficiency using MPA. Twenty-two biological pathways were significantly altered, with the top 10 including aminoacyl-tRNA biosynthesis; purine metabolism; phenylpropanoid biosynthesis; pyrimidine metabolism; arginine and proline metabolism; glutathione metabolism; tryptophan metabolism; diterpenoid biosynthesis; glycerophospholipid metabolism; alanine, aspartate, and glutamate metabolism; and linolenic acid metabolism (Table 1).

Metabolic analysis suggested that MYB30 may be involved in antioxidant regulation under iron deficiency. Iron deficiency stress led to ROS generation, which plants counteract via antioxidant defense systems (Gill and Tuteja, 2010; Song et al., 2022). Therefore, we examined the accumulation of ROS in seedlings under iron deficiency by histochemical staining with DAB. Compared to Col-0, myb30 plants showed more intense DAB staining, whereas MYB30-OX plants exhibited weaker staining, indicating higher and lower ROS accumulation, respectively, under iron-deficient conditions (Figure 6A, lower panel). Under normal growth conditions, there were no differences in H₂O₂ levels in the tested seedlings by quantitative measurements. However, under iron deficiency, myb30 plants showed higher accumulation of H₂O₂, whereas MYB30-OX plants exhibited lower accumulation of H₂O₂, when compared to the level in WT plants (Figure 6B). These outcomes revealed that myb30 plants experienced greater oxidative stress under iron deficiency.

Enzymatic antioxidants are crucial for ROS scavenging. Therefore, the activities of CAT, SOD, and POD in seedlings were assessed. When grown on iron-deficient conditions, all genotypes exhibited reduced antioxidant enzyme activity, with myb30 mutants showing the lowest activity and MYB30-OX lines showing the highest activity (Figure 7). Correspondingly, higher antioxidant enzyme activity was associated with lower H₂O₂ accumulation in transgenic plants. RT-qPCR analysis revealed that the expression of SOD, POD, and CAT was significantly lower in myb30 plants and higher in MYB30-OX plants compared to WT under iron deficiency (Figure 8). These results suggested that MYB30 reduces ROS accumulation by upregulating ROS-associated genes and enhancing antioxidant enzyme activity during iron deficiency stress.

Iron uptake capacity is impaired under iron deficiency, affecting iron homeostasis (Forieri et al., 2017). We measured iron concentrations in seedlings subjected to iron deficiency and found that iron accumulation was significantly reduced in all plants, with myb30 mutants showing lower iron concentrations than WT (Supplementary Figure 2). We also analyzed the expression of key iron uptake and translocation genes, including NAS4, PYE, FRO2, FIT, IRT1, and ZIF1 (Colangelo and Guerinot, 2004; Palmer et al., 2013; Brumbarova et al., 2015). Under iron deficiency, the expression of NAS4, FRO2, and IRT1 was significantly lower in myb30 mutants compared to WT (Figure 9). These results suggest that MYB30 orchestrates the expression of genes involved in iron uptake and translocation, thereby enhancing iron accumulation, which is positively associated with enhanced tolerance to iron deficiency.