Overcoming Mn-induced chlorosis in sugarcane seedlings by iron

Background: Manganese (Mn) toxicity induces severe seedling chlorosis and growth inhibition in sugarcane cultivated on acidic soils, yet the mechanisms driving seasonal recovery and scalable mitigation strategies remain poorly defined. This study aimed to elucidate the role of iron (Fe) deposition from rainwater in natural greening and validate foliar Fe supplementation as an efficient countermeasure.

Methods: We integrated field monitoring across 78 sites, phenological tracking of seasonal recovery, molecular analyses of chlorophyll biosynthesis pathways, and validation experiments (hydroponic and field trials) to investigate Fe-mediated Mn toxicity alleviation. Key metrics included leaf chlorophyll/Fe correlations, gene expression patterns, and agronomic responses to Fe treatments.

Results: Field data revealed a strong positive correlation between leaf chlorophyll content and foliar Fe levels (r=0.82, p<0.01). Chlorotic seedlings achieved full visual recovery by late summer, with chlorophyll and Fe concentrations increasing 11.1- and 4.4-fold relative to spring baselines. Mechanistically, Fe reversed Mn-induced functional Fe deficiency by enhancing 5-aminolevulinic acid synthesis (2.3-fold increase) and Mg-protoporphyrin IX monomethyl ester conversion (1.8-fold increase), while downregulating FLUORESCENT expression (60% reduction) and upregulating MgPME cyclase activity (3.1-fold increase). Foliar FeSO₄ applications (0.5–1.5 g Fe L⁻¹) effectively reversed chlorosis, boosting chlorophyll content by 1.9–2.7-fold, seedling survival by 100%, and cane yield by 1.7-fold under Mn-toxic conditions, with minimal input requirements (7.5–22.5 g Fe ha⁻¹).

Conclusion: Our findings demonstrate that rainwater-borne Fe is a key driver of seasonal recovery from Mn-induced chlorosis in sugarcane. Foliar Fe supplementation emerges as a cost-effective, scalable strategy for mitigating Mn toxicity, offering significant advantages over resource-intensive soil amendments for sustainable sugarcane production on acidic soils.

Introduction

Soil acidification is a major global constraint to agricultural productivity, with acidic soils (pH ≤ 5.5) occupying over 50% of the world’s arable land, particularly in tropical and subtropical regions (Wang et al., 2023). In China, approximately 32% of the total land area is acidic, mainly in the south (Zhao et al., 2023). Since the 1980s, soil pH in major crop-production regions has declined by 0.13–0.80 units, with more severe acidification observed in the leached red and yellow soils of southern China (Guo et al., 2010; Wang et al., 2023). When soil pH drops below 5.5, divalent manganese ions (Mn²⁺) are released into the soil solution, becoming a major constraint to plant growth and crop production (Horst, 2012; Barker and Pilbeam, 2015). Excessive Mn interferes with the uptake, translocation, and utilization of other essential elements, inhibits enzyme activity, induces oxidative stress, and ultimately reduces photosynthesis and plant growth (Barker and Pilbeam, 2015; Zhu et al., 2025). Mn toxicity causes visible leaf chlorosis in maize (Zea mays L.) (Stoyanova et al., 2009), bush bean (Phaseolus vulgaris L.) (Horiguchi, 1988), and barley (Hordeum vulgare L.) (Huang et al., 2018), and is associated with chlorophyll (Chl) breakdown due to photobleaching and/or photooxidative damage to chloroplasts in common bean (Huang et al., 2018; Gonzalez et al., 1998), sugar maple (Acer saccharum Marsh.), and red maple (Acer rubrum L.) (St Clair and Lynch, 2004).

Iron (Fe), the most abundant transition metal in soils, commonly coexists with Mn in soil minerals and becomes more soluble under acidic conditions. The well-documented antagonistic interaction between Fe and Mn arises from their similar ionic radii, competition for shared transport systems, analogous chemistry, and partially overlapping physiological functions (Barker and Pilbeam, 2015; Shao et al., 2017; Yang et al., 2022). Previous studies have shown that Fe supply can mitigate Mn toxicity by reducing tissue Mn accumulation in Arabidopsis (Arabidopsis thaliana L.) (Yang et al., 2022) and soybean (Glycine max L.) (Van Der Vorm and Van Diest, 1979), restoring Mn-impaired Fe metabolism in soybean (Yi et al., 2022), alleviating Mn-induced chlorosis in barley (Huang et al., 2016), soybean, and sunflower (Helianthus annuus L.) (Mehrotra and Gupta, 1990; Shen et al., 2017), and enhancing Mn tolerance in Arabidopsis (Yang et al., 2022). However, Fe–Mn interactions vary among species. In barley and tomato (Solanum lycopersicum L.), Fe supply does not significantly alter tissue Mn concentrations (Yi et al., 2022; Foy et al., 1998), and in rice (Oryza sativa L.) Fe application has no effect on Mn uptake (Van Der Vorm and Van Diest, 1979). These contrasting responses may reflect differences in experimental conditions (e.g., pH, Fe/Mn ratios) or species-specific adaptations in Mn homeostasis and detoxification.

Sugarcane (Saccharum spp. hybrids), an important tropical crop for sugar, fiber, and biofuel production (Yi et al., 2022), is often grown on acidic soils where Mn toxicity frequently causes severe leaf chlorosis and markedly reduces yield (Yang et al., 2022; Huang et al., 2016). Our previous mechanistic studies indicate that Mn toxicity in sugarcane primarily impairs chlorophyll biosynthesis through dual inhibition of ALA synthesis and MgPME conversion, rather than by accelerating chlorophyll degradation (Yang et al., 2022). Field observations also show that chlorotic seedlings can recover greenness during late-summer rainfall, suggesting an environment-dependent recovery mechanism. This study therefore aimed to (1) elucidate the physiological mechanisms underlying rain-associated greening in Mn-stressed sugarcane, (2) determine the role of Fe in this process, and (3) assess whether these mechanisms can inform the development of highly effective agronomic strategies to mitigate Mn-induced chlorosis under field conditions. We hypothesized that rainwater-derived Fe facilitates seasonal recovery by restoring chlorophyll biosynthesis, and that foliar Fe supplementation can replicate this effect under field conditions.

Materials and methods

Field surveys

To assess whether Mn-induced chlorosis in sugarcane is associated with foliar Fe nutrition, field surveys were conducted during March–April 2014 in six major sugarcane-producing regions of Guangxi, China (Chongzuo, Laibin, Nanning, Liuzhou, Yizhou, and Guigang), located within 22˚33ˊ-22˚43ˊN latitude and 107˚31ˊ-109°24ˊE longitude, which together account for over 50% of national sugarcane cultivation. Soils in these areas are strongly acidic (pH 3.6–4.8) and consistently induce seedling-stage chlorosis (Martínez-Cuenca et al., 2013). In each of 78 cultivation plots, the first fully expanded leaves of sugarcane cv. XTT22 were collected from 30-day-old seedlings. From each plot, 20 plants were randomly selected, and their leaves were pooled to form one composite sample, with three biological replicates per plot (total n = 234). Samples were placed in ice-cooled insulated containers and transported to the laboratory for Fe and chlorophyll (Chl) analyses.

Seasonal changes in leaf Fe concentration during chlorosis recovery were monitored in three geographically separated plots in Quli Town, Fusui County (22˚59N, 107˚584ˊE), Guangxi, in 2016. Severely chlorotic seedlings (SPAD < 4) were sampled at six time points: day 0 (April 1), 5, 15, 25, 35, and 60 after identification. The first fully expanded leaves were analyzed for active Fe and Mn concentrations, and non-destructive Chl measurements were taken from 20 randomly selected plants per plot using a SPAD-502 Plus meter (Konica Minolta, Japan).

Culture experiments

A hydroponic experiment was conducted to evaluate the role of rainwater in alleviating Mn-induced chlorosis. Ratoon seedlings (cv. Guitang 32) were regenerated from parent plants pre-treated with 0.5 mM MnCl₂ in one-fifth-strength Hoagland solution (pH 5.5) for 30 days under controlled growth chamber conditions (28°C, 16/8 h light/dark). Chlorotic seedlings (15 days old) were transplanted into 5.5-L plastic pots (4 seedlings per pot) containing aerated Fe-free one-fifth-strength Hoagland solution prepared with either filtered rainwater or deionized water (control), with or without foliar spraying (three times daily) of the corresponding water. Rainwater was collected in May 2018 using acid-washed polyethylene containers. The collected rainwater exhibited the following basic properties: pH 5.91, Fe (0.26 mg L⁻¹), Ca (0.74 mg L⁻¹), Mg (0.03 mg L⁻¹), K (0.11 mg L⁻¹), NO₃^- (0.77 mg L⁻¹), SO₄²⁻ (1.30 mg L⁻¹), and Cl⁻ (0.17 mg L⁻¹). After 15 days, the first fully expanded leaves were photographed, analyzed for Chl concentration (SPAD-502 meter), and harvested for Fe and Mn determination.

To examine the effect of Fe supplementation, seedlings (cv. Guitang 32) prepared as described by Yang (Yang et al., 2022) were cultivated in 5.5-L plastic pots (4 seedlings per pot) and exposed to one of three treatments based on an Fe-free one-fifth-strength Hoagland solution (pH 5.5): (1) control (Fe-free solution), (2) 0.5 mM Mn (MnCl₂), and (3) 0.5 mM Mn + 10 μM Fe (supplied with EDTA-Fe). Fifteen days after treatment, the first fully expanded leaves were analyzed for Chl, 5-aminolevulinic acid (ALA), Mg-protoporphyrin IX monomethyl ester (MgPME), Mg-protoporphyrin IX (MgProto), protochlorophyllide (Pchlide), and expression levels of FLUORESCENT (FLU) and MgPME cyclase (MgPMEC).

Field experiment

Field trials were conducted in 2024 at a sugarcane plantation (22°41′N, 107°48′E) located in Quli Town, Chongzuo City, Guangxi, China, on acidic lateritic soil with naturally high Mn bioavailability. The soil was developed from Quaternary red clay parent material. It was characterized by the following basic properties: pH 5.1, organic matter content of 18.78 g kg⁻¹, total nitrogen content of 1.14 g kg⁻¹, available phosphorus content of 41.0 mg kg⁻¹, available potassium content of 193.0 mg kg⁻¹, and available manganese content of 321.0 mg kg⁻¹. Sugarcane (cv. Guitang 32) was planted in 2023 at a density of 105,000 buds per hectare. Similar experiments were also conducted during the 2022 and 2023 growing seasons at three additional field sites, and the 2024 trial is presented here as a representative example. These trials employed a randomized complete block design with plots each covering an area of 100 m² and three biological replicates per treatment. On May 9, seedlings exhibiting severe Mn toxicity (interveinal chlorosis, high leaf Mn concentration, and reduced Chl concentration) were foliar-sprayed with FeSO₄ solution at four Fe concentrations: 0 (control), 0.5, 1.0, or 1.5 g L⁻¹. The spray was applied at a rate of 750 kg ha^-1. Leaf appearance was documented at 0, 3, 13, and 33 days after treatment (DAT). The first fully expanded leaves were sampled for Fe and Chl analyses, and seedling survival was recorded at 13 DAT. At maturity, cane yield was measured through plot-wide stalk harvesting, weighing, and yield calculation.

Determination of Fe and Mn concentrations

Fe and Mn concentrations were measured by flame atomic absorption spectrometry (PinAAcle 900T, PerkinElmer) (Yang et al., 2022). Fresh leaves (~1 mm fragments) were prepared using stainless steel scissors; dried leaves were oven-dried at 70°C to constant weight, ground, digested with concentrated HNO₃ at 140°C, and analyzed for total Fe and Mn (Yang et al., 2022). Rainwater Fe concentration was measured after filtration. Active Fe in fresh leaves was extracted by immersion in 1 M HCl for 24 h with occasional shaking, filtered, and quantified spectrophotometrically at 510 nm (UV2600, Shimadzu, Japan) using ortho-phenanthroline as the chelating agent (Mehrotra and Gupta, 1990).

Determination of chlorophyll concentration

Chl was extracted from fresh leaves with 80% (v/v) acetone in the dark until complete bleaching. Absorbance was measured at 645 and 663 nm (UV2600, Shimadzu, Japan), and total Chl concentration was calculated following Yang et al. (2022).

Determination of Proto, MgProto, and Pchlide

Proto, MgProto, and Pchlide were determined as described by Yang et al. (2022). Fresh leaves were ground in liquid nitrogen, extracted in 80% alkaline acetone (v/v, alkalized with 0.1 M NH₄OH), and incubated in the dark until bleaching. After centrifugation (15,000 ×g, 10 min, 4°C), absorbance of the supernatant was measured at 575, 590, and 628 nm, and pigment concentrations were calculated as previously described (Shen et al., 2017).

Gene expression analysis

Upon termination of the experiments, the first expanded leaves were collected, immediately frozen in liquid nitrogen, and stored at -80°C until analysis. Total RNA was extracted from the leaves, followed by cDNA synthesis and quantitative real-time PCR (qRT-PCR); gene expression levels were then determined using gene-specific primers, all according to the methods described by Yang et al. (2022). The β-actin gene was used as a reference gene for gene expression data normalization.

Statistical analysis

Results were analyzed by using analysis of variance. Where the F-test indicated significant differences (P ≤ 0.05), treatment means were compared by Duncan’s multiple range test. All analyses and graphical presentations were performed using Origin Pro 2019.

Results

Relationship between leaf Fe and chlorophyll concentration

Mn-induced chlorosis in sugarcane seedlings grown on acidic soils was confirmed in Guangxi, China (Supplementary Figure 1A). During the dry spring, affected seedlings exhibited interveinal chlorosis; however, surviving plants progressively regained greenness, with complete symptom remission by the rainy summer season (Supplementary Figures 1B, C). Across five major production cities, a strong positive correlation was observed between leaf Fe and chlorophyll (Chl) concentrations (r = 0.82, p < 0.01; Figure 1). Elevated leaf Fe concentrations were consistently associated with higher Chl levels under Mn stress, suggesting a role for Fe in mitigating Mn-induced chlorosis.

Figure 1

Figure 1. Relationship between leaf iron (Fe) concentration and chlorophyll content in sugarcane seedlings grown on acidic soils (pH 3.2–5.5). Samples were collected from 78 independent field plots during March–April. Each point represents a composite sample of 20 pooled leaves, with three biological replicates per plot. Values are means ± SE (n = 3). ** indicate a significant Pearson correlation (p<0.01).

Temporal dynamics of leaf Fe during chlorosis recovery

In 2016 field monitoring plots, natural recovery of chlorotic seedlings began in mid-May, with full greenness restored in newly emerged leaves by early June. Mn concentrations in all tissues exceeded established toxicity thresholds (Barker and Pilbeam, 2015), with mean values of 767.5 ± 9.9 mg kg⁻¹ DW (leaf blade), 295.1 ± 4.5 mg kg⁻¹ DW (leaf sheath), and 624.7 ± 17.9 mg kg⁻¹ DW (stem) (Figure 2A). Leaf SPAD values increased by 71% at 35 days after observation (DAO) and by 4.4-fold at 60 DAO relative to chlorotic baselines (Figure 2B), coinciding with complete symptom disappearance (SPAD > 48.1 ± 3.7). Leaf Fe concentration increased 0.7-fold at 35 DAO and 4.4-fold at 60 DAO, supporting the association between Fe accumulation and chlorosis recovery.

Figure 2

Figure 2. Seasonal changes in (A) leaf Fe concentration and (B) SPAD values in sugarcane seedlings grown on strongly acidic soil. Leaves were sampled on April 1 (day 0) and subsequently at the indicated time points. Values are means ± SE (n = 3). Significant differences in (A) leaf Fe concentration among tissue types were determined by one-way ANOVA, while differences in (B) SPAD values across sampling dates were analyzed by repeated-measures one-way ANOVA. For analyses showing significant overall effects, post hoc comparisons were performed using Duncan’s test. Prior to ANOVA, SPAD data were subjected to arcsine transformation. Different lowercase letters indicate statistically significant differences (p < 0.05).

Rainwater alleviates Mn-induced chlorosis

Long-term field observations (since 2010) showed that chlorotic seedlings recovered following the onset of the rainy season. Under controlled conditions, rainwater application—via both foliar spray and root-zone exposure—restored greenness within 15 days (Figure 3A), increasing SPAD values by 2.7-fold compared with baseline (p < 0.01; Figure 3B). In contrast, deionized water controls developed progressive albinism with no SPAD change (Figures 3A, B). These results demonstrate that rainwater is sufficient to trigger greening in Mn-stressed sugarcane seedlings.

Figure 3

Figure 3. Effects of rainwater application on Mn-induced chlorotic sugarcane seedlings. (A) representative leaves at 15 days after treatment (DAT); (B) SPAD values at 0 and 15 DAT; (C) leaf Fe and Mn concentrations at 15 DAT. Values are means ± SE (n = 3). **above the bars indicate a significant difference at p<0.01 (Student’s t-test).

Rainwater supplies bioavailable Fe to chlorotic leaves

Rainwater application increased leaf Fe concentration by 1.4-fold compared with the deionized water control (p < 0.01; Figure 3C), without affecting Mn concentration. Rainwater contained an average of 0.26 mg L⁻¹ Fe, indicating a direct Fe supply to chlorotic leaves. The lack of recovery in Fe-free controls confirms that rainwater-derived Fe is essential for alleviating Mn-induced chlorosis.

Fe restores chlorophyll biosynthesis in Mn-stressed seedlings

Hydroponic experiments showed that Fe supplementation (Mn+Fe) increased leaf Fe concentration by 1.1-fold and Chl concentration by 1.9-fold relative to Mn-only seedlings (Figure 4A). Chl levels in Mn+Fe plants were comparable to controls (Con), indicating full reversal of Mn-induced chlorosis.

Figure 4

Figure 4. Effects of Fe supplementation on concentrations of (A) leaf Fe and chlorophyll, (B) chlorophyll precursors, and (C) relative expression of Flu and MgPMEC. Seedlings were grown in nutrient solution under three treatments: control (Con), excess Mn²⁺ (0.5 mmol L⁻¹, Mn), and excess Mn²⁺ plus Fe (10 μmol L⁻¹, Mn+Fe) for 15 days. Values are means ± SE (n = 3). Different lowercase letters indicate significant differences (p < 0.05), as determined by one-way ANOVA with Duncan’s multiple range test (the same for all following figures unless otherwise stated).

Mn toxicity inhibited chlorophyll biosynthesis by reducing 5-aminolevulinic acid (ALA) and downstream intermediates, and by causing Mg-protoporphyrin IX monomethyl ester (MgPME) accumulation (Yang et al., 2022. Fe supplementation increased ALA by 1.0-fold, Mg-protoporphyrin IX (MgProto) by 1.9-fold, and protochlorophyllide (Pchlide) by 2.0-fold, while decreasing MgPME by 23.6% (Figure 4B). ALA was fully restored to control levels; MgProto and Pchlide recovered to >90% of control levels.

Mn stress suppressed MgPME cyclase (MgPMEC) expression and upregulated FLUORESCENT (Flu), which encodes an inhibitor of ALA biosynthesis. Fe supplementation restored MgPMEC expression to control levels and reduced Flu expression by 85% relative to Mn-only plants (Figure 4C).

Foliar Fe application alleviates Mn-induced chlorosis in field-grown seedlings

Foliar FeSO₄ sprays (0.5-1.5 g Fe L⁻¹) were applied to chlorotic seedlings (leaf Mn concentration: 360.4 ± 4.9 mg kg⁻¹ DW) grown in acidic soil (pH 5.1) in 2024. Similar trials were conducted in 2022–2023 at three additional sites, and the 2024 results are presented as representative. Three days after application, chlorosis symptoms visibly diminished; by 13 days after treatment (DAT), leaves were largely green in Fe-treated plants, whereas control seedlings developed severe chlorosis (Figure 5). By 33 DAT, most control plants exhibited extensive chlorosis and necrosis, with some mortality, while Fe-treated plants maintained vigorous growth with darker green leaves, greater leaf number, and increased height.

Figure 5

Figure 5. Visual recovery of Mn-induced chlorotic sugarcane seedlings after foliar application with 0.5 g L⁻¹ Fe (as FeSO₄ solutions) at (A) 0, (B) 3, (C) 13, and (D) 33 DAT in acidic soil (pH 5.1).

Leaf active Fe and Chl concentrations increased with Fe spray concentration at 13 DAT (Figure 6), and Chl levels continued to rise until 33 DAT (Figure 7). Foliar Fe application also improved agronomic performance: survival plant number increased (Figure 8A), and cane yield reached 100.3-116.3 t ha⁻¹, 2.68–3.11 times higher than controls (0 g Fe L⁻¹), with the highest yield at 1.0 g Fe L⁻¹ (Figure 8B). These results demonstrate that foliar Fe application is an effective field-level strategy to mitigate Mn-induced chlorosis and enhance sugarcane yield in acidic soils.

Figure 6

Figure 6. Effects of foliar FeSO₄ application on (A) leaf Fe concentration and (B) chlorophyll content in Mn-induced chlorotic sugarcane seedlings at 13 DAT. Plants were sprayed with FeSO₄ solutions containing 0, 0.5, 1.0, or 1.5 g L⁻¹ Fe. Values are means ± SE (n = 3).

Figure 7

Figure 7. Time course of leaf chlorophyll content in Mn-induced chlorotic sugarcane seedlings following foliar application of FeSO₄ solutions containing 0.5 g L⁻¹ Fe. Measurements were taken at 0, 3, 13, and 33 DAT. Values are means ± SE (n = 3). Different lowercase letters indicate significant differences, as determined by two-way ANOVA by Duncan’s multiple range test (p < 0.05).

Figure 8

Figure 8. Effects of foliar FeSO₄ application on (A) number of surviving seedlings at 13 DAT and (B) mature cane yield in Mn-induced chlorotic sugarcane. Plants were sprayed with FeSO₄ solution containing 0, 0.5, 1.0, or 1.5 g L⁻¹ Fe. Values are means ± SE (n = 3).

Discussion

Seasonal recovery of Mn-induced chlorosis in sugarcane seedlings is facilitated by iron derived from rainwater

Previous studies have identified Mn toxicity as the primary cause of widespread chlorosis in ratoon sugarcane seedlings grown on acidic soils in China. However, the developmental progression of chlorosis and its underlying recovery mechanisms remain poorly understood. In this study, we confirmed Mn-induced chlorosis during the dry spring, followed by progressive greening in surviving seedlings and complete symptom remission during the rainy summer. Mechanistic evidence demonstrated that this recovery was facilitated by Fe deposition from rainwater, which counteracted Mn-induced functional Fe deficiency and alleviated the inhibitory effects of Mn toxicity on chlorophyll biosynthesis.

Mn-induced Fe deficiency is a recognized mechanism of Mn phytotoxicity in plants (Foy et al., 1998; Kabir et al., 2016), and visual symptoms can resemble those of Fe deficiency in species such as Stylosanthes, pineapple, and sunflower (Blamey et al., 2019; Chen et al., 2015). In sugarcane seedlings, Mn toxicity causes uniform chlorosis in the veins and mesophyll of emerging leaves, with older leaves exhibiting yellowing and whitening (Horiguchi, 1988; Ling et al., 2022). Across 78 surveyed sites, leaf chlorophyll and Fe concentrations were strongly correlated (R = 0.82, p < 0.01; Figure 1). Phenological monitoring further showed that chlorotic seedlings accumulated up to 10-fold more foliar Fe by late summer than in early spring (Figure 2), coinciding with sharp increases in precipitation (Supplementary Figure 2). This seasonal recovery is consistent with reports that chlorotic ratoon sugarcane plants tend to exhibit reduced Mn concentrations after spring, likely due to a dilution effect (Huang et al., 2016). Rainwater application under controlled conditions induced visible greening and increased leaf chlorophyll by 2.7-fold (Figure 3B), whereas controls showed progressive albinism. Soil Fe availability remained unchanged (data not shown, p > 0.05), while foliar Fe deposition increased 4.4-fold (Figure 2B), suggesting rainwater as the primary Fe source for recovery. The rainwater also contained elements such as N, Mg, Ca, and S. Previous studies have ruled out N, S, or Mg deficiency as causes of sugarcane seedling chlorosis in acidic soils (Huang et al., 2016), supporting the view that Fe is central to Mn-induced chlorosis. However, other seasonal factors such as temperature, light availability, and canopy development also varied alongside seedling greening, and whether these factors contribute independently or interactively to recovery remains to be addressed. Together, these observations suggest that Fe supply plays an important role in the recovery process of chlorotic seedlings.

The biochemical basis of this recovery aligns with our previous findings that Mn-induced chlorosis results from impaired chlorophyll biosynthesis—specifically, inhibition of δ-aminolevulinic acid (ALA) synthesis and reduced conversion of magnesium-protoporphyrin IX monomethyl ester (MgPME) (Yang et al., 2022). Here, Mn stress decreased ALA, MgProto, and Pchlide, while increasing MgPME accumulation (Figure 4B), alongside upregulation of the ALA biosynthesis inhibitor FLUORESCENT (FLU) and downregulation of MgPMEC (Figure 4C). Fe supplementation restored ALA to control levels, reduced MgPME accumulation, and upregulated MgPMEC, while suppressing Flu expression by 85%. These results suggest that rainwater-derived Fe facilitates the reactivation of ALA synthesis and relieves the MgPME bottleneck, thus restoration of chlorophyll biosynthesis and potentially enabling complete recovery of chlorotic seedlings by late summer.

Foliar Fe application as a field-level strategy to alleviate Mn-induced chlorosis

Although Fe supply in growth media is known to reduce Mn uptake and toxicity (Barker and Pilbeam, 2015; Alam et al., 2001; Martínez-Cuenca et al., 2013), field-based evidence for the efficacy of foliar Fe against Mn phytotoxicity has been limited. Our trials provide direct field confirmation that a single foliar Fe spray can rapidly and effectively reverse Mn-induced chlorosis in sugarcane. Visible improvement occurred within 3 days, with complete greenness by 13 days after treatment (DAT) (Figure 5). This was accompanied by significant increases in leaf Fe and chlorophyll concentrations (Figures 6, 7), with chlorophyll accumulation showing both dose- and time-dependence, peaking at 2.1 ± 0.1 mg g⁻¹ FW at 33 DAT with 1.0 g Fe L⁻¹.

Yield recovery under Mn stress has been rarely reported for foliar Fe fertilization (Horst, 2012; Martínez-Cuenca et al., 2013; Barker and Pilbeam, 2015; Huang et al., 2015; Kabir et al., 2016). In this study, a single foliar FeSO₄ application fully restored yield to levels comparable to healthy plants, achieving up to a 3.11-fold increase over Mn-toxicity controls. The treatment provided sustained benefits throughout the growth cycle, despite the limited phloem mobility typically associated with foliar Fe (Horst, 2012; Barker and Pilbeam, 2015), suggesting that foliar Fe may be more effective against Mn toxicity than against Fe deficiency alone.

Effective Fe spray concentrations vary widely among species, from 0.01 g L⁻¹ in strawberry to 1.8 g L⁻¹ in pear, pepper, and groundnut (Xiao et al., 2003; Pestana et al., 2012; Roosta and Mohsenian, 2012). In sugarcane, leaf Fe and chlorophyll concentrations increased with Fe spray concentrations from 0.5 to 1.5 g L⁻¹; however, survival and final yield did not differ significantly among treatments, indicating that 0.5 g L⁻¹ (equivalent to 7.5 g Fe ha⁻¹) is sufficient for Mn toxicity mitigation. This foliar approach requires far lower Fe inputs than soil amendments, such as FeSO₄ applications at 4.5–200.1 kg ha⁻¹ for peanut, pepper, and blueberry (Xiao et al., 2003; Roosta and Mohsenian, 2012; Pu et al., 2019), or CaCO₃ incorporation at up to 5 kg per tree for Mn-toxic woody species (Chatzistathis et al., 2015). These findings highlight foliar Fe application as a resource-efficient, scalable strategy for alleviating Mn-induced chlorosis and improving sugarcane productivity on acidic soils, with substantial advantages in Fe use efficiency and cost-effectiveness over conventional soil treatments.

Conclusion

Our findings suggest that Fe deposition from rainwater contributes to the seasonal recovery of Mn-induced chlorosis in sugarcane by alleviating Mn-induced Fe deficiency and subsequently reactivating chlorophyll biosynthesis. Importantly, foliar Fe application (optimal at 0.5 g L⁻¹) provides an effective mitigation strategy that restores both leaf chlorophyll content and crop yield under field conditions. This study establishes rainwater Fe as a natural remediation pathway but also proposes foliar Fe supplementation as a scalable solution for Mn toxicity in acidic soils.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Author contributions

DL: Investigation, Writing – review & editing, Formal Analysis, Data curation. GL: Investigation, Methodology, Data curation, Writing – original draft. SY: Methodology, Conceptualization, Supervision, Funding acquisition, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This study was supported in part by the National Natural Science Foundation of China (32360451), Guangxi Science and Technology Base and Talent Special Project (AD25069107), and the National Natural Science Foundation of China (32460799).

Acknowledgments

We extend our sincere gratitude to the students of our research group for their meticulous work in the analysis and processing of soil and plant samples.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1739211/full#supplementary-material

Supplementary Figure 1 | Sequential recovery of Mn-induced chlorotic sugarcane seedlings in a commercial field on (A) April 18, (B) June 11, and (C) July 3, 2013.

Supplementary Figure 2 | Cumulative daily precipitation from April 1 to June 30, 2018, in the study area.

References

Alam, S., Kamei, S., and Kawai, S. (2001). Amelioration of manganese toxicity in barley with iron. J. Plant Nutr. 24, 1421–1433. doi: 10.1081/PLN-100106992

Crossref Full Text | Google Scholar

Barker, A. V. and Pilbeam, D. J. (2015). Handbook of plant nutrition (New York: CRC Press, Taylor & Francis Group).

Google Scholar

Blamey, F. P. C., Li, C., Howard, D. L., Cheng, M. M., Tang, C. X., Scheckel, K. G., et al. (2019). Evaluating effects of iron on manganese toxicity in soybean and sunflower using synchrotron-based x-ray fluorescence microscopy and x-ray absorption spectroscopy. Metallomics 11, 2097–2110. doi: 10.1039/c9mt00219g

PubMed Abstract | Crossref Full Text | Google Scholar

Chatzistathis, T., Alifragis, D., and Papaioannou, A. (2015). The influence of liming on soil chemical properties and on the alleviation of manganese and copper toxicity in juglans regia, robinia pseudoacacia, eucalyptus sp. and populus sp. Plantations. J. Environ. Manage 150, 149–156. doi: 10.1016/j.jenvman.2014.11.020

PubMed Abstract | Crossref Full Text | Google Scholar

Chen, Z. J., Sun L, L., Liu, P. D., Liu, G. D., Tian, J., and Liao, H. (2015). Malate synthesis and secretion mediated by a manganese-enhanced malate dehydrogenase confers superior manganese tolerance in stylosanthes guianensis. Plant Physiol. 167, 176–178. doi: 10.1104/pp.114.251017

PubMed Abstract | Crossref Full Text | Google Scholar

Foy, C. D., Farina, M. P. W., and Oakes, A. J. (1998). Iron-manganese interactions among clones of nilegrass. J. Plant Nutr. 21, 987–1009. doi: 10.1080/01904169809365459

Crossref Full Text | Google Scholar

Gonzalez, A., Steffen, K. L., and Lynch, J. P. (1998). Light and excess manganese: implications for oxidative stress in common bean. Plant Physiol. 118, 493–504. doi: 10.1104/pp.118.2.493

PubMed Abstract | Crossref Full Text | Google Scholar

Guo, J. H., Liu, X. J., Zhang, Y., Shen, J. L., Han, W. X., Zhang, W. F., et al. (2010). Significant acidification in major Chinese croplands. Science 5968, 1008–1010. doi: 10.1126/science.1182570

PubMed Abstract | Crossref Full Text | Google Scholar

Horiguchi, T. (1988). Mechanism of manganese toxicity and tolerance of plants VII. Effect of light intensity on manganese-induced chlorosis. J. Plant Nutr. 11, 235–246. doi: 10.1080/01904168809363799

Crossref Full Text | Google Scholar

Horst, M. (2012). Marschner’s mineral nutrition of higher plants. 3rd Edn (Beijing: Academic Press).

Google Scholar

Huang, X., Fan, Y., Shabala, L., Rengel, Z., Shabala, S., and Zhou, M. X. (2018). A major QTL controlling the tolerance to manganese toxicity in barley (Hordeum vulgare L.). Mol. Breed. 38, 16. doi: 10.1007/s11032-017-0767-9

Crossref Full Text | Google Scholar

Huang, Y. L., Lan, L. Y., Hu, S. H., Zhang, C. L., Feng, J. L., Tang, J., et al. (2015). Impact of organic fertilizers and iron-fertilizers on Morus alba L. chlorosis in paddy soils. Acta Agric. Zhejiangensis 27, 1984–1989. doi: 10.3969/j.issn.1004-1524.2015.11.20

Crossref Full Text | Google Scholar

Huang, Y. L., Yang, S., Long, G. X., Zhao, Z. K., Li, X. F., and Gu, M. H. (2016). Manganese toxicity in sugarcane plantlets grown on acidic soils of southern China. PloS One 11, e148956. doi: 10.1371/journal.pone.0148956

PubMed Abstract | Crossref Full Text | Google Scholar

Kabir, A. H., Paltridge, N., and Stangoulis, J. (2016). Chlorosis correction and agronomic biofortification in field peas through foliar application of iron fertilizers under Fe deficiency. J. Plant Interact. 11, 1–4. doi: 10.1080/17429145.2015.1125534

Crossref Full Text | Google Scholar

Ling, G. Z., Xiao, J. L., Yang, S., Li, D. L., Tang, X. L., Wang, X. X., et al. (2022). The alleviation of manganese toxicity by ammonium in sugarcane is related to pectin content, pectin methyl esterification, and nitric oxide. GCB Bioenergy 14, 585–596. doi: 10.1111/gcbb.12936

Crossref Full Text | Google Scholar

Martínez-Cuenca, M. R., Quiñones, A., Iglesias, D. J., Forner-Giner, MÁ., Primo-Millo, E., and Legaz, F. (2013). Effects of high levels of zinc and manganese ions on strategy in responses to Fe deficiency in citrus. Plant Soil 373, 943–953. doi: 10.1007/s11104-013-1861-3

Crossref Full Text | Google Scholar

Mehrotra, S. C. and Gupta, P. (1990). Reduction of iron by leaf extracts and its significance for the assay of Fe (II) iron in plants. Plant Physiol. 93, 1017–1020. doi: 10.1104/pp.93.3.1017

PubMed Abstract | Crossref Full Text | Google Scholar

Pestana, M., Correia, P. J., Saavedra, T., Gama, F., Abadía, A., and de Varennes, A. (2012). Development and recovery of iron deficiency by iron resupply to roots or leaves of strawberry plants. Plant Physiol. Biochem. 53, 182–191. doi: 10.1016/j.plaphy.2012.01.001

PubMed Abstract | Crossref Full Text | Google Scholar

Pu, S. Y., Yang, J. H., Ji, X. N., Xia, K. L., Song, K., and Wang, Z. X. (2019). Effects of different types of iron fertilizer and fertilization on the photosynthetic pigment content of blueberry leaves. J. Tianjin Agric. Univ. 26, 6–9. doi: 10.19640/j.cnki.jtau.2019.01.002

Crossref Full Text | Google Scholar

Roosta, H. R. and Mohsenian, Y. (2012). Effects of foliar spray of different Fe sources on pepper (Capsicum annum L.) plants in aquaponic system. Sci. Hortic. 146, 182–191. doi: 10.1016/j.scienta.2012.08.018

Crossref Full Text | Google Scholar

Shen, Y., Li, J. F., Gu, R. C., Yue, L., Zhan, X. H., and Xing, B. S. (2017). Phenanthrene-triggered chlorosis is caused by elevated chlorophyll degradation and moisture. Environ. pollut. 220, 1311–1321. doi: 10.1016/j.envpol.2016.11.003

PubMed Abstract | Crossref Full Text | Google Scholar

Shao, J. F., Yamaji, N., Shen, R. F., and Ma, J. F. (2017). The key to Mn homeostasis in plants: Regulation of Mn transporters. Trends Plant Sci. 22, 215–224. doi: 10.1016/j.tplants.2016.12.005

PubMed Abstract | Crossref Full Text | Google Scholar

St Clair, S. B. and Lynch, J. P. (2004). Photosynthetic and antioxidant enzyme responses of sugar maple and red maple seedlings to excess manganese in contrasting light environments. Funct. Plant Biol. 31, 1005. doi: 10.1071/FP04049

PubMed Abstract | Crossref Full Text | Google Scholar

Stoyanova, Z., Poschenrieder, C., Tzvetkova, N., and Doncheva, S. (2009). Characterization of the tolerance to excess manganese in four maize varieties. Soil Sci. Plant Nutr. 55, 747–753. doi: 10.1111/j.1747-0765.2009.00416.x

Crossref Full Text | Google Scholar

Van Der Vorm, P. D. J. and Van Diest, A. (1979). Aspects of the Fe and Mn nutrition of rice plants I. Iron- and manganese uptake by rice plants, grown under aerobic and anaerobic conditions. Plant Soil 51, 233–246. doi: 10.1007/BF02232886

Crossref Full Text | Google Scholar

Wang, P., Cao, H. R., Quan, S. X., Wang, Y., Li, M., Wei, M., et al. (2023). Nitrate improves aluminium resistance through SLAH-mediated citrate exudation from roots. Plant Cell Enviro. 46, 3518–3541. doi: 10.1111/pce.14688

PubMed Abstract | Crossref Full Text | Google Scholar

Xiao, Y., Li, Y. T., and Cao, Y. P. (2003). Effect of Fe-fertilizer composition and application methods on the iron chlorosis correction of peanut. Soil Feit. China 28, 21–25.

Google Scholar

Yang, S., Ling, G., Li, Q., Yi, K., Tang, X., Zhang, M., et al. (2022). Manganese toxicity-induced chlorosis in sugarcane seedlings involves inhibition of chlorophyll biosynthesis. Crop J. 10, 1674–1682. doi: 10.1016/j.cj.2022.04.008

Crossref Full Text | Google Scholar

Yi, K., Li, X. F., Chen, D. W., Yang, S., Liu, Y., Tang, X. L., et al. (2022). Shallower root spatial distribution induced by phosphorus deficiency contributes to topsoil foraging and low phosphorus adaption in sugarcane (saccharum officinarum L.). Front. Plant Sci. 12, 797635. doi: 10.3389/fpls.2021.797635

PubMed Abstract | Crossref Full Text | Google Scholar

Zhao, X., Pan, X., Ma, H., Dong, X., Che, J., Wang, C., et al. (2023). Scientific issues and strategies of acid soil use in China. Acta Pedol. Sin. 60, 1248–1263. doi: 10.11766/trxb202307250290

Crossref Full Text | Google Scholar

Zhu, H. Y., Pan, J. C., Wei, Y. Y., Lan, H. Y., Yang, S., Li, X. F., et al. (2025). Manganese toxicity suppressing nitrogen-fixing bacteria growth and impairing nitrogen uptake and utilization in sugarcane. Front. Microbiol. 16. doi: 10.3389/fmicb.2025.1548896

PubMed Abstract | Crossref Full Text | Google Scholar