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. 2021 May 6;28(8):4770–4777. doi: 10.1016/j.sjbs.2021.04.092

Early-stage iron deficiency alters physiological processes and iron transporter expression, along with photosynthetic and oxidative damage to sorghum

Sadia Akter Prity a, Ahmed M El-Shehawi b, Mona M Elseehy c, Sharaban Tahura a, Ahmad Humayan Kabir a,⁎
PMCID: PMC8324970  PMID: 34354465

Abstract

Iron (Fe) starvation in Strategy II plants is a major nutritional problem causing severe visual symptoms and yield reductions. This prompted us to investigate the physiological and molecular consequences of Fe deficiency responses at an early stage in sorghum plants. The Fe-starved sorghum did not show shoot biomass reduction, but the root length, biomass, and chlorophyll synthesis were severely affected. The chlorophyll a fluorescence analysis showed that the quantum yield efficiency of PSII (Fv/Fm) and photosynthesis performance index (Pi_ABS) in young leaves significantly reduced in response to low Fe. Besides, Fe concentration in root and shoot significantly declined in Fe-starved plants relative to Fe-sufficient plants. Accordingly, this Fe reduction in tissues was accompanied by a marked decrease in PS-release in roots. The qPCR experiment showed the downregulation of SbDMAS2 (deoxymugineic acid synthase 2), SbNAS3 (nicotianamine synthase 3), and SbYSL1 (Fe-phytosiderophore transporter yellow stripe 1) in Fe-deprived roots, suggesting that decreased rhizosphere mobilization of Fe(III)-PS contributes to reduced uptake and long-distance transport of Fe. The cis-acting elements of these gene promoters are commonly responsive to abscisic acid and methyl jasmonate, while SbYSL1 additionally responsive to salicylic acid. Further, antioxidant defense either through metabolites or antioxidant enzymes is not efficient in counteracting oxidative damage in Fe-deprived sorghum. These findings may be beneficial for the improvement of sorghum genotypes sensitive to Fe-deficiency through breeding or transgenic approaches.

Keywords: Fe deficiency, PS release, Oxidative injury, Strategy II plants

1. Introduction

Iron (Fe) deficiency in soil having bicarbonate, high pH, and ferric Fe is a major nutritional disorder in plants (Alcántara et al., 2000). These soil conditions affect the bioavailability of Fe for plants and thus, affect plant growth and yield. As a co-factor, Fe functions in several cellular processes, which include photosynthesis, respiration, and protein formation in plants (Zhang et al., 2019). Photosystem II (PS-II) contains Fe proteins and mislays its efficiency in Fe-deprived plants because of low photosynthetic electron supply (Jiang et al., 2007, Bertamini et al., 2002). As a result, Fe deficiency causes stunned root, leaf chlorosis, and poor maturation in plants (Kabir et al., 2013, Wang et al., 2007).

Sorghum (Sorghum bicolor) as a Strategy-II plant possesses a chelation-based Fe uptake system. In the rhizosphere, Strategy-II plants discharge inorganic phytosiderophores (PS) to form Fe-PS complexes resulted in the mobilization of inorganic Fe‐III (Vert et al., 2002). Among the chelating molecules associated with Fe-uptake, 2′-deoxymugineic acid (DMA) and nicotianamine synthases (NAS) are the most common in Strategy II plants (Pearce et al., 2014). DMA-related chelation of insoluble Fe increases the solubility and absorption of Fe(III)-DMA in the root system. Barley and wheat secrete a considerable amount of DMA under Fe deficiency (Römheld and Marschner, 1990). The NAS converts three molecules of S-adenosyl methionine into NA, which is a Fe chelator involved in Fe transport in plants (Inoue et al., 2003; Higuchi et al., 1995). In other Strategy II plants, OsDMAS1 and HvDMAS1 genes have been identified to play roles in DMA synthesis (Kobayashi et al., 2010, Bashir et al., 2006). In addition, higher plants use NAS in the plant cell for chelation and maintenance of Fe homeostasis (Hell and Stephan, 2003). In a transgenic study, tobacco was introduced with the barley HvNAS1 gene ultimately showed increased Fe and Zn in the shoot (Takahashi et al., 2003). Also, the YSL (yellow stripe-like transporter) gene acts as a root transporter of the chelator Fe3+-PS complex (Yordem et al., 2011, Curie et al., 2001).

Fe-deficiency contributes to over-accumulation of reactive oxygen species (ROS), leading to oxidative damages in plant cells (Kabir et al., 2015). However, several antioxidant metabolites and enzymes are often in action to regulate the redox balance, although which is mostly seen in Fe-efficient plant species/cultivars (Kabir et al., 2015, Yang et al., 2015, Ashraf, 2009). Moreover, antioxidant activities in response to stress varied among the plant species, tissues, and subcellular localization (Mittova et al., 2003).

Sorghum is a popular cereal for its importance as grain food, residual crop, and animal feed. Only a few studies reported the responses of Fe-deprived sorghum, but those were solely limited to genotype screening (Obour et al., 2019, Clark et al., 2008). Understanding the responses or consequences of damage due to Fe-deficiency may provide useful knowledge in improving the survivability and yield of sorghum through agronomic or biotechnological approaches. However, Fe-deficiency adversely affects sorghum plants is not yet fully understood. Therefore, we investigated how Fe deficiency lessens growth and development in sorghum plants. Along with the morpho-physiological characterization, a broad range of cellular and molecular responses were studied to determine the mechanistic basis of growth retardation in Fe-deficient sorghum.

2. Materials and methods

2.1. Plant cultivation

The surface of sorghum seeds (var. sucrosorgo 405) was sterilized for 5 min with 70 percent ethanol before placing on moist tissue paper for 3d in a tray at 28 °C. The germinated seedlings were then transferred to solution culture (pH 6.0) supplemented with nutrients (Hoagland and Arnon 1950) as follows (µM): KNO3 (1600), Ca(NO3)2·4H2O (6 0 0), KH2PO4 (1 0 0), MgSO4·7H2O (2 0 0), KCl (50), H3BO3 (25), MnSO4·4H2O (2), Na2MoO4·2H2O (0.5) and CuSO4·5H2O (0.5). The Fe was supplemented as follows: +Fe (25 µM Fe-EDTA) and -Fe (1.0 µM Fe-EDTA). In each plastic container (4L), nine plants were kept at 25 °C with 60% relative and a light intensity of 200 μmolm−2 s−1 under long-day conditions (14 h light/ 10 h dark) in the growth chamber. The nutrients were replaced every 4d. The plants were cultivated for 14 d before data analysis.

2.2. Characterization of morphological and photosynthetic features

The longest root and shoot were measured by a digital caliper. The dry weight of root and shoot was taken after drying for 3d at 80 °C in an electric oven. The chlorophyll score was measured on young leaves by SPAD meter (Minolta, Japan). Chlorophyll a fluorescence kinetics (OJIP) such as Fv / Fm (photosystem II quantitative efficiency) and Pi ABS (photosynthesis index), were also measured by FluorPen 100 (photon systems instruments, Czech Republic) in dark-adapted young leaves for 1 h.

2.3. Analysis of Fe concentration

Tomato root specimens were washed once with 0.1 mM CaSO4 and rinsed multiple times with Milli-Q water to remove the surface contaminants. Roots and shoots were dried separately in the oven at 70 °C for 72 h. Dry samples were then weighed and dissolved with HClO4/HNO3, 1:3 v/v. The concentration of Fe in the digestion solution was determined by atomic absorption spectroscopy (AA-6800, Shimadzu, Japan).

2.4. Determination of the release of PS in roots

The PS secretion in roots was analyzed as described earlier (Khobra and Singh, 2018). Briefly, entire plants were submerged in a pump-aerated beaker containing 1 N NaOH and 4 mM FeCl3 for 3 h. The solution was then filtered through Whatman filter paper (grade: 1, diameter: 125 mm, Merck, India) to eliminate the degradation of the microbes. The Fe(III) was then reduced by adding 8 percent hydroxylaminochloride at 55 °C for 20 min. Subsequently, 10 mM ferrozine and 0.5 M Na-acetate buffer (pH 4.6) were added prior to absorbance monitoring of the solution (Fe3+ ion) at 562 nm.

2.5. Expression pattern of candidate genes and promoter analysis

The total RNA in roots was extracted using SV RNA extraction system (Promega, USA). The quantified RNA was then converted to cDNA using the reverse-transcription system before PCR analysis in an Eco™ real-time PCR system (Illumina, USA) using gene-specific primers for SbDMAS2, SbNAS3 and SbYSL1 genes (Supplementary Table S1). The PCR reactions were set as follows: 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 sec, 57 °C for 30 sec. The relative expression of candidate genes was calculated by the dd − ΔCt method (Livak and Schmittgen, 2001) considered Actin as an internal control. The qPCR experiment was repeated three times. Besides, the PlantCare web tool was used for scanning the cis-elements present in promoter regions of SbDMAS2, SbNAS3 and SbYSL1 promoters (Lescot et al., 2002).

2.6. Analysis of stress indicators

Bovine serum albumin (BSA) curve was plotted to estimate the total soluble protein by Bradford assay (Guy et al., 1992). In short, fresh root and shoot sample protein extraction were performed by grinding with Tris-HCl buffer (50 mM, pH 7.5), 0.04% (v / v) β-mercaptoethanol, and 2 mM EDTA. The crude samples were centrifuged at 12000 rpm for 10 min before collecting the transparent fluid portion. Finally, 1 ml of Coomassie Brilliant Blue (CBB) was added to the supernatant before measuring the absorbance at 595 nm.

The loss of cell membrane integrity was measured by a conductivity meter in both root and shoot (Lutts et al., 1996). Briefly, root and shoot surface components were washed with deionized water. The fresh specimens were then transferred to a deionized water beaker (20 ml) and incubated at 25 °C for 2 h. Afterward, the solution's electrical conductivity (EC1) was calculated. The samples were then heated in a water bath for 20 min at 95 °C to ensure the maximum release of electrolytes and then cooled down to 25 °C. The final EC (EC2) was then recorded and determined as follows: = (EC1/EC2) × 100 (%).

Evans blue method was used to determine the rate of cell death in the root and shoot (Zhao et al., 2005). The entire fresh root and shoot were transferred 2 ml of Evan's blue mixture and waited 15 min. The suspension was subsequently dried up to 10 min in 1 ml of 80% ethanol. The tubes with solutions were then incubated in a water bath for 15 min at 50 °C and then centrifuged for 10 min at 12000 rpm. The absorbance of the supernatant was measured at 600 nm. Finally, the percentage of cell death was evaluated based on sample fresh weight.

2.7. Estimation of H2O2 and O2–

The root and shoot samples were homogenized in 0.1% trichloroacetic acid (Alexieva et al., 2001). The extracts were then centrifuged at 10,000 rpm for 15 min. The top aqueous segment was mixed with 10 mM potassium phosphate (pH 7.0), 1 M KI, and placed in a dark room to allow reaction for 1 h. Finally, the absorbance of the solution was read at 390 nm. Besides, the superoxide (•O2−) was calculated using a coefficient of extinction 2.16 × 104 M−1·cm−1), as described by Hu et al. (2012). Briefly, the fresh samples were washed with water, homogenized with chilled K-phosphate buffer (10 mM), and centrifuged at 12000 rpm at 4 °C for 10 min. The clear supernatant was mixed with an assay solution containing 0.5 mM XTT sodium salt and 50 mM Tris-HCl (pH 7.5). Finally, the solution's optical density was read at 580 nm.

2.8. Analysis of antioxidant enzymes

Briefly, plant root and shoot were independently homogenized in mortar pestle with 100 mM phosphate buffer (pH 7.0). The homogenate was centrifuged for 10 min at 8000 rpm, and the transparent part was collected for further analysis. The 100 µL plant extract was added with 0.1 mM EDTA, 50 mM NaHCO3 (pH 9.8), and 0.6 mM epinephrine for the determination of SOD activity (Goud and Kachole, 2012). After 4 min, the confirmation of adrenochrome was read at 475 nm. For ascorbate peroxidase (APX) activity, 0.1 mM EDTA, 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM H2O2, and 0.5 mM ascorbic acid were added to 100 µL extract (Sun and Zigman, 1978). Afterward, the absorbance was recorded at 290 nm and APX activity was calculated based on the extinction coefficient (2.8 mM−1 cm−1). The catalase (CAT) activity was analyzed by mixing 100 µL plant extract with 100 mM MKP-buffer (adjusted pH 7.0), 6% hydrogen peroxide. Finally, the absorption of the solution was reported at 240 nm in 30 sec to 1 min interval (extinction coefficient 0.036 mM−1 cm−1). In addition, 100 μL of extract was added to 0.2 M KP-buffer (pH 7.0), 1 mM EDTA, 0.2 mM NADPH and 20 mM oxidized glutathione (GSSG) for (glutathione reductase) GR analysis. The absorbance of the solution was read at 340 nm. Finally, the GR activity was calculated using the extinction coefficient (6.12 mM−1 cm−1) as described earlier (Halliwell and Foyer, 1978).

2.9. Analysis of S-metabolites

Briefly, plant samples were washed with ultra-pure water and then dried in an oven. The samples were pulverized using a milling machine into a fine powder. The samples were then dissolved with 50% methanol and filtered using Whatman filter paper (grade: 1, diameter: 125 mm, Merck, India). We analyzed the amino acid in root and shoot with dual-wavelength (280 and 360 nm) by Waters 2489 high-performance liquid chromatography (HPLC) in C18 reverse-phase column (Kabir et al., 2016). We maintained gradient conditions (pore size: 300 A, particle size: 5 μm, pH Range: 1.5–10, Dimension: 250 mm × 10 mm; column oven temperature: 250 °C, flow rate: 1.0 ml/min, run time: 45 min) using 100% acetonitrile as the mobile phase (Kabir et al., 2016). The samples were diluted (100 × ) and then filtered (0.22 μm Minisart Syringe Filters) before injection (20 µL).

2.10. Statistical analysis

In this study, each experiment was repeated three times for consistency in plants cultivated in a randomized block design. The significance of the mean of replications was assessed by t-test using Microsoft Excel 2007 at a 5% significance level. In preparing graphical figures, GraphPad Prism 6 was used.

3. Results

3.1. Plant growth, photosynthesis efficiency, and Fe concentration

Fe starvation induced a significant reduction in root length and dry weight in contrast to Fe-sufficient controls (Fig. 1a–c). However, shoot height and dry weight did not show any significant changes following Fe shortage compared to Fe-sufficient plants (Fig. 1a–c). The SPAD score, an indicator of chlorophyll synthesis, was significantly reduced in young leaves under Fe starvation than in the controls (Fig. 2a). Further, the Fv/Fm value of the Fe-sufficient sorghum was found to be above 0.8, while Fe-starved plants showed a significant decline in Fv/Fm (below 0.6) value (Fig. 2b). Similarly, Pi_ABS value showed a significant reduction under Fe-deficiency in young leaves compared to plants cultivated with sufficient Fe (Fig. 2c).

Fig. 1.

Fig. 1

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Plant phenotype (a), tissue length (b) and dry weight (c) of 14d-old sorghum plants cultivated in Fe-sufficient and Fe-deficient conditions. Different letters in Fig. (c) and (d) indicate significant differences between means ± SD of treatments (n = 3) at a P < 0.05 significance level.

Fig. 2.

Fig. 2

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SPAD score (a), Fv/Fm (b) and Pi_ABS (c) in leaves of 14d-old sorghum plants in Fe-sufficient and Fe-deficient conditions. Different letters indicate significant differences between means ± SD of treatments (n = 3) at a P < 0.05 significance level.

3.2. Fe concentration, PS release and gene expression

The root and shoot Fe concentration of sorghum plants decreased considerably under low Fe supplement compared to plants cultivated with sufficient Fe in the hydroponic culture (Fig. 3a). PS release from the roots remarkably decreased in Fe-starved sorghum in contrast to Fe-adequate plants (Fig. 3b). We have analyzed the relative transcript levels in the roots of the sorghum of key Fe-acquisition genes. Plants exposed to Fe starvation exhibited a significant downregulation of SbDMAS1, SbNAS1, and SbYSL1 genes in roots relative to Fe-adequate conditions (Fig. 3c).

Fig. 3.

Fig. 3

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Tissue Fe concentration (a), PS release (b) and expression of candidate genes (c) in roots of 14d-old sorghum plants cultivated in Fe-sufficient and Fe-deficient conditions in 14d-old sorghum plants. Different letters indicate significant differences between means ± SD of treatments (n = 3) at a P < 0.05 significance level.

The significant cis-acting elements in these gene promoters were ABRE (cis-acting element involved in the abscisic acid responsiveness), CGTCA-motif (cis-acting regulatory element involved in the methyl jasmonate-responsiveness), G-Box (cis-acting regulatory element involved in light responsiveness), O2-site (cis-acting regulatory element involved in zein metabolism regulation), TGACG-motif (cis-acting regulatory element involved in the methyl jasmonate -responsiveness), GCN4_motif (cis-regulatory element involved in endosperm expression), LTR (cis-acting element involved in low-temperature responsiveness), motif I (cis-acting regulatory element root-specific), AuxRR-core (cis-acting regulatory element involved in auxin responsiveness), CAT-box (cis-acting regulatory element related to meristem expression), MSA-like (cis-acting element involved in cell cycle regulation), TCA-element (cis-acting element involved in salicylic acid responsiveness) as analyzed by PlantCare (Table 1).

Table 1.

Cis-regulatory element analysis of SbDMAS2, SbNAS3 and SbYSL1 gene promoters.

Gene promoters ABRE CAAT-box CGTCA-motif G-Box O2-site TGACG-motif GCN4_motif LTR motif I AuxRR-core CAT-box MSA-like TCA-element
SbDMAS2 3 4 3 4 1 3
SbNAS3 4 2 4 6 4 1 1 1
SbYSL1 5 8 4 6 1 2 2 1 1 1
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ABRE (cis-acting element involved in the abscisic acid responsiveness), CAAT-box (common cis-acting element in promoter and enhancer regions), CGTCA-motif (cis-acting regulatory element involved in the methyl jasmonate -responsiveness), G-Box (cis-acting regulatory element involved in light responsiveness), O2-site (cis-acting regulatory element involved in zein metabolism regulation), TGACG-motif (cis-acting regulatory element involved in the methyl jasmonate -responsiveness), GCN4_motif (cis-regulatory element involved in endosperm expression), LTR (cis-acting element involved in low-temperature responsiveness), motif I (cis-acting regulatory element root specific), AuxRR-core (cis-acting regulatory element involved in auxin responsiveness), CAT-box (cis-acting regulatory element related to meristem expression), MSA-like (cis-acting element involved in cell cycle regulation), TCA-element (cis-acting element involved in salicylic acid responsiveness)

3.3. Changes in stress indicators

Fe deficiency showed a significant decline in total soluble protein in root and shoot of sorghum relative to Fe-sufficient plants (Fig. 4a). In addition, electrolyte leakage in roots increased dramatically in contrast to controls due to Fe starvation. However, this phenomenon did not vary in the shoot of sorghum between Fe adequate and Fe-deficient conditions (Fig. 4b). Besides, cell death (%) in both root and shoot remarkably increased following Fe-deficiency in contrast to Fe-sufficient controls (Fig. 4c).

Fig. 4.

Fig. 4

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Changes in different stress indicators in root and shoot of sorghum cultivated in Fe-sufficient and Fe-deficient conditions in 14d-old sorghum plants. Different letters in each column indicate significant differences between means ± SD of treatments (n = 3) at a P < 0.05 significance level.

3.4. Reactive oxygen species

Compared to Fe-adequate plants, the H2O2 concentration was significantly induced in the roots of Fe-starved sorghum (Fig. 5a). However, Fe-starvation caused no significant changes in H2O2 concentration in the shoot in comparison with Fe-sufficient controls (Fig. 5a). Further, the reactive O2– in root and shoot significantly increased due to Fe-deficiency relative to Fe-sufficient plants (Fig. 5b).

Fig. 5.

Fig. 5

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Changes in ROS molecules and different antioxidant enzymes in root and shoot of sorghum cultivated in Fe-sufficient and Fe-deficient conditions in 14d-old sorghum plants. Different letters indicate significant differences between means ± SD of treatments (n = 3) at a P < 0.05 significance level.

3.5. Antioxidant enzymes

In this study, the CAT and SOD activities showed a substantial decline in the root but not in the shoot in Fe-deprived plants relative to controls (Fig. 5c and d). Nevertheless, Fe-shortage did not show any effect on APX and GR activities in root and shoot subjected to Fe-shortage in contrast to Fe-adequate plants (Fig. 5e and f).

3.6. Relative changes in S-metabolites

In this study, cysteine and methionine significantly declined in the root, although none of the amino acids did vary in shoot under Fe-starvation in contrast to Fe-sufficient plants (Fig. 6a and b). However, glutathione concentration in root or shoot did not vary following Fe deficiency relative to Fe-sufficient conditions (Fig. 6c).

Fig. 6.

Fig. 6

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Changes in S-metabolites in root and shoot of sorghum cultivated in Fe-sufficient and Fe-deficient conditions in 14d-old sorghum plants. Different letters indicate significant differences between means ± SD of treatments (n = 3) at a P < 0.05 significance level.

4. Discussion

4.1. Plant growth and photosynthesis under Fe deficiency

This study explores our understanding of how Fe-deficient sorghum plants respond at an early stage. Induction of Fe deficiency caused severe growth retardation in the roots, although the shoot biomass and length were not affected. It may be possible that the shoot was not affected due to a short growth period, which is also observed in sorghum previously (Hirai et al., 2007). Along with the visual leaf symptoms, the SPAD score dramatically dropped due to Fe starvation, suggesting the occurrence of photosynthesis efficiency in sorghum leaves. To further confirm it, Chl a fluorescence analysis consistently showed the decrease in Fv/Fm value in leaves. It actively supports the diagnosis of Fe-deficiency symptoms and suggests the reduction in quantum yield efficiency in Fe-deprived sorghum. The decrease in the quantum yield of PSII is often associated with the Fe-deficient leaves (Abadía et al., 1999). Further, Pi_ABS was also consistently declined in Fe-deprived leaf of sorghum, which might be linked to the low energy conservation efficiency index by PS-II antenna. In C4 plants, mesophyll and bundle sheath cells are mostly affected by Fe deficiency (Stocking, 1975). However, PSI is more sensitive than PSII as it contains several Fe atoms. As PSI activity gets arrested, the PSII center functions, adjusting the reduced efficiency of PSI to transport the electrons to ferredoxin (Sharma, 2007). Several studies documented that a chlorotic leaf is closely correlated with PSII efficacy in Fe-deprived plants (Gogorcena et al., 2001, Donnini et al., 2003). However, this relation may vary depending on the plant species and mineral deficiency. Under Mn deficiency, barley plants showed a decrease in PSII activity, although the chlorophyll was stable in leaves (Schmidt et al., 2013). Our findings indicate that Fe-starvation triggers the changes in PS-II activity that is directly related to the reaction center or various energy transfer pathways in sorghum leaves. This message can be useful to strengthen the knowledge to avoid damage to the photosynthetic apparatus in Fe-starved sorghum.

4.2. Changes in Fe concentration and transporter genes

The Fv/Fm value near 0.8 in healthy plants is usually considered safe for non-stress conditions. Hence, chlorophyll a fluorescence is a popular strategy to study photosynthetic systems in stress-induced plants (Baker and Rosenqvist, 2004). In this study, the relationship between quantum yield efficiency and tissue Fe was further evaluated. The Fe concentration was severely decreased under Fe starvation in sorghum. Although the root morphology is consistent with root Fe concentration, the shoot biomass did not change owing to Fe starvation. However, the chlorophyll and PSII status clearly states that Fe-deficiency does not appear to inhibit the development of aerial parts but is closely linked to photosynthetic kinetics in sorghum.

Alternation in Fe uptake, which is another spotlight of the study, was accompanied by the decreased release of PS in Fe-starved sorghum. PS possesses a chelation affinity for Fe(III) in Strategy II plants, allowing high Fe flow into the roots (Inoue et al., 2009, Curie et al., 2001). In this study, we noticed a substantial decrease of PS release in roots following Fe-deficiency in sorghum. Khobra and Singh (2018) demonstrated that Fe-efficiency was accompanied by higher PS release relative to the wheat lines inefficient to cope with Fe-deprivation (Khobra and Singh, 2018). The release of PS is dependent on plant Fe status as Fe is involved with the deactivation or activation of organic ligands (Mimmo et al., 2014).

We also studied the expression of transporter genes typically found in plants within Strategy II relating to Fe uptake and PS release under Fe-deficiency. In this study, the substantial decrease of SbDMAS2, SbNAS3, and SbYSL1 in Fe-deprived roots implies that Fe uptake and bioavailability in the rhizosphere were severely affected by Fe shortage resulting in stunted growth and physiological damages in sorghum plants. These results on the transporter genes in sorghum are contradictory to a few Strategy II plant species. In wheat, DMAS and YSL homologs were also up-regulated on iron hunger (Wang et al., 2019). However, it is known that sorghum exhibits different substrate specificity and expression patterns of transporters than barley and maize subjected to Fe deficiency (Hirai et al., 2007). In rice, NA transporters were induced under iron deficiency in the root epidermis (Inoue et al., 2008). It is widely known that YSL transporters are associated with the transport of Fe from root to shoot in Strategy II plants (Koike et al., 2004, Curie et al., 2001). In barley, HvNAS1 and HvTOM1 genes sharply induced after Fe deficiency resulted in Fe utilization and redistribution within the shoot (Nikolic et al., 2019). However, based on our finding, it appears that Fe availability in the rhizosphere is largely affected due to the downregulation of SbDMAS2, SbNAS3, and SbYSL1-mediated decreased transportation of Fe to the aerial part in Fe-starved sorghum. This coincides with chlorosis and PS-II damage in Fe-starved sorghum. It is also known that strategies to overcome Fe-deficiency symptoms are highly genotype-specific. Besides, promoter analysis reveals the involvement of cis-acting elements associated with several stress-responsive elements, such as abscisic acid, methyl jasmonate and salicylic acid. Studies reported that methyl jasmonate signaling is involved in the expression of Fe-deficiency induced genes in plants (Kobayashi et al., 2016), while salicylic acid signaling trigger transcriptional regulation of downstream Fe-related genes (Zuchi et al., 2015).

4.3. Redox status and antioxidant properties

Photo-oxidative damage is often observed in Fe-deficient plants. Plants starving with Fe are more prone to oxidative stress as Fe is a co-factor of many antioxidant enzymes. In this study, none of the S-metabolites were found to be induced due to Fe shortage in sorghum, indicating that the Fe status of the plant may also be linked to the S status of the sorghum plants. In maize, Fe deficiency caused excess Zn and thiol accumulation along with growth reduction (Kanai et al., et al., 2009). S-induced physiological and antioxidant activities are commonly seen in Fe-starved plants (Zuchi et al., 2009). In plants, chloroplast and photosystem machinery is closely linked with the involvement of Fe-S proteins (Forieri et al., 2013, Vigani et al., 2009). Additionally, many enzymes involved in S assimilation include Fe. Besides, the accumulation of S is frequently related to the accumulation of Met, the forerunner of PS (Luna et al., 2018). Further, CAT and SOD activity severely affected following Fe deficiency in sorghum. The relation of Fe shortage with decreased SOD activity in Arabidopsis was reported (Ramírez et al., 2013). Whether Fe deficiency impairs ROS balance in Strategy II plants is still not adequately studied. However, some evidence has been published on the association of low Fe availability and reduced activities of SOD and APX in sunflower and Arabidopsis (Ramírez et al., 2013, Ranieri et al., 2000). However, APX and GR showed no induction in sorghum tissues subjected to Fe-starvation, suggesting that inefficiency to mitigate excess ROS is possibly related to the damage of chloroplasts and cellular proteins. However, the ROS regulation in plant cells may vary on the species and cultivar of species.

5. Conclusion

This research offers essential knowledge on the mechanistic basis of Fe-starvation in sorghum. Fe shortage caused stunned growth, photosynthetic inefficiency, and cellular damage in sorghum. The substantial decrease of Fe in tissues was mostly dependent on PS-mediated Fe mobilization accompanied by the downregulation of PS-related genes in sorghum roots. Besides, in silico analysis revealed the presence of cis-regulatory elements in promoters associated with several stress-responsive elements, such as methyl jasmonate and salicylic acid. Furthermore, antioxidant defense neither by S-metabolites or antioxidant enzymes AMF assisted Fe-deprived sorghum plants in overcoming oxidizing injuries. These results provide the necessary context for reducing Fe-deficiency symptoms in sorghum or other Strategy II plants.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Acknowledgment

We are thankful to the Central Science Laboratory of Rajshahi University.

Funding statement

The current work was funded by Taif University Researchers Supporting Project number (TURSP - 2020/75), Taif University, Taif, Saudi Arabia.

Footnotes

Peer review under responsibility of King Saud University.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.sjbs.2021.04.092.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (15.2KB, docx)

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