Iron (Fe) is an essential micronutrient, participating in various vital processes in cell metabolism due to its redox status between ferrous (Fe²⁺) and ferric (Fe³⁺) forms. Fe functions as an electron acceptor or donor actively participating in photosynthesis and respiration (Kobayashi and Nishizawa, 2012; Zhai et al., 2014). Lowland and flooded soil are poor in oxygen concentration and become acidic. The flooding of rice fields leads to the reduction of Fe³⁺ to Fe²⁺, which becomes highly toxic to paddy crops. Fe²⁺ toxicity is a major cause of abiotic stress affecting large rice-growing areas, especially in Asian countries (Becker and Asch, 2005). Depending on stress intensities, a loss of 12% to 100% has been reported on rice yields (Sahrawat, 2005; Audebert and Fofana, 2009). Fe²⁺ toxicity is a severe agricultural constrain that generally occurs in acidic soils. Thus, understanding the underlying molecular mechanisms associated with their adaptation may help to build strategies for future rice breeding programs for Fe²⁺ toxicity tolerance.

Angiosperms plants have evolved two acquisition strategies to overcome the limited Fe availability viz., strategy I and II. The strategy I is based on the acidification of rhizospheres by releasing protons from the roots, which reduces Fe³⁺ to Fe²⁺ and its transportation through iron-regulated transporter (IRT) across the root plasma membrane. Strategy II is based on the uptake of Fe³⁺ phytosiderophore complexes. Although being a Strategy II plant, rice (Oryza sativa L.) possesses Fe²⁺ transporter genes (OsIRT1 and OsIRT2) that can directly uptake Fe²⁺ from the soil (Bughio, 2002; Ishimaru et al., 2006). The mechanism of Fe²⁺ toxicity tolerance in rice plants has been highlighted in several studies (Asch et al., 2005; Deng et al., 2010; Wu et al., 2017; Aung et al., 2018; Aung and Masuda, 2020). Rice plants possess several defense mechanisms to cope with Fe²⁺ toxicity. The defense mechanism of rice plants for Fe²⁺ tolerance can be divided into four mechanisms (Aung and Masuda, 2020). Defense 1 (Fe exclusion from roots) is a root-based tolerance mechanism where Fe plaques (Fe³⁺ precipitation) on the root surface acts as a barrier to the uptake of Fe²⁺ into root tissues. Fe plaque is formed due to the rhizospheric oxidation of Fe²⁺ to Fe³⁺ by oxygen transport from shoots to roots (Asch et al., 2005; Becker and Asch, 2005). Defense 2 (Fe retention in roots and suppression of Fe translocation to shoots) where discrimination center and ferritin (OsFER) plays an important role by retaining excess Fe in root and avoiding translocation into the shoot. Defense 3 (Fe compartmentalization in shoots) includes compartmentalization, disposal, or storage of Fe inside shoots by vacuolar iron transporter (OsVIT1/OsVIT2), and OsFER1/2. Defense 4 [reactive oxygen species (ROS) detoxification] includes enzymatic detoxification, scavenging of ROS by antioxidants. Defense 1–3 was highlighted to work on mild to moderate Fe excess conditions and do not seriously affect rice growth. In contrast, defense 4 was hypothesized to work at a molecular level under Fe severe conditions that causes bronzing and inhibit plant growth and development (Aung and Masuda, 2020). ROS antioxidants like glutathione S-transferase (GST) and ascorbate oxidase were reported to play an important role in shoot-based Fe tolerance in rice plants (Wu et al., 2017). Besides, under severe Fe excess conditions, cytochrome P450 family proteins, OsNAC4, OsNAC5, and OsNAC6, played an important role in alleviating ROS (Aung and Masuda, 2020). The elucidation of the genes involved in response to Fe-toxicity is fundamental to understanding the mechanism that confers tolerance to stress and the development of tolerant cultivars. With the advancement of next generation sequencing (NGS) technology, the RNA-seq technique has become a valuable tool for transcriptional profile analysis, providing a better understanding of gene responses to Fe²⁺ toxicity. To date, many studies on the transcriptional profile of rice under Fe-stress have been done. However, there is still a broad spectrum that needs better clarification to understand the Fe stress mechanism, which can help to understand the unsolved queries related to Fe toxicity in rice.

In this study, the transcriptome of aromatic rice was analyzed in both root and leaves using RNA-Seq. Along with the common Fe-homeostasis-related genes, the study identified several differentially expressed genes (DEGs) related to the ROS scavenging system. The results provided a comprehensive transcriptome resource that may enrich the understanding of Fe-homeostasis under Fe²⁺ toxicity conditions.

Fe²⁺ toxicity significantly inhibits the growth and productivity of rice. Fe²⁺ toxicity interrupts the metabolism and functioning of plants, affecting root and shoot development (Aung et al., 2018; Regon et al., 2021). A previous study reported the toxic effect of 2.5 mM Fe²⁺ in aromatic Keteki Joha (Regon et al., 2021). The present study performed a transcriptomic analysis of Keteki Joha under severe Fe²⁺ toxicity (2.5 mM) to investigate how it responds under stress conditions. Fe homeostasis genes were well characterized in Fe deficient condition; however, the molecular mechanism of Fe toxicity in rice plants is still not well empathized (Bashir et al., 2011; Kobayashi et al., 2014). In this study, nearly 69 Fe homeostasis-related genes and their homologs were identified, which were mostly downregulated. The current study shares similarities with the previous microarray result with several other exceptions (Finatto et al., 2015; Aung et al., 2018). Thus, these findings will help to further ameliorate the understanding of Fe homeostasis in rice. Differential exon usages of few Fe homeostasis genes were identified, from which possible alternative splicing of DEGs can be inferred (Anders et al., 2012). However, validation of the predicted genes may be essential to conclude any transcriptional alteration or changes under Fe²⁺ toxicity.

Four different types of tolerance mechanisms have been reported in rice (Aung and Masuda, 2020). However, Fe sensing is crucial at the initial response stage in Fe homeostasis gene expression and regulation based on Fe availability (Kobayashi and Nishizawa, 2014). OsIDEF1, OsIDEF2, and OsIRO2 positively regulate Fe deficiency-inducible genes involved in DMA-based Fe²⁺ acquisition, Fe²⁺ uptake and translocation. In addition, the OsIDEF1 is directly bound with Fe²⁺ and other divalent metal ions, suggesting an intracellular Fe sensor (Kobayashi, 2019). Moreover, OsHRZ1 and OsHRZ2 can also bind with Fe via hemerythrin domains, thus characterized as another Fe sensor candidate. Their knockouts have negatively modulated the response to Fe-deficiency in rice. In rice, OsIDEF1 controls the expression of both OsHRZ1 and OsHRZ2 (Li et al., 2019a). However, in this study, the OsIDEF1 gene was not differentially expressed among all the tissues. OsHRZ1 being downregulated, suppression of Fe transporter genes at transcriptional level are quite unlike under severe Fe²⁺ conditions. Besides, OsHORZ1, a haemerythrin domain-containing protein, was also downregulated, which is known to repress OsHRZ functions (Kobayashi and Nishizawa, 2014, 2015; Kobayashi, 2019). In this context, further study of transcription factors in contrasting genotypes may provide a better understanding of their downstream regulation and proper functioning under Fe²⁺ toxicity. However, Fe-excess responses in rice plants are thought to be partially independent of Fe-deficiency (Kobayashi and Nishizawa, 2014). A positive Fe-deficiency regulator OsPRI1, a target gene of OsHRZ1 was also unexpressed in this study. Despite that, OsNAS3 was upregulated, which is important for Fe detoxification in the root (Aung et al., 2019). In addition, OsFRDL1 was also upregulated, which is responsible for root-to-shoot Fe translocation and is believed to play a significant role in the distribution of Fe into old leaves. Thus, OsFRDL1 is crucial for the minimization of Fe²⁺ toxicity in roots. As a result, OsFER1/OsFER2 and OsVIT2 were also highly upregulated in leaves, thus defending the Fe overload. The current study is reasonably supported by the proposed Fe-excess defense mechanisms (Aung and Masuda, 2020). In exception, OsVMT was highly upregulated in both root and leaves, and it is known to be expressed where Fe and Zn are highly deposited. Knockout of these genes enhances Fe and Zn accumulation in polished rice grains as DMA increases solubilization of Fe and Zn deposited in the node (Che et al., 2019). Considering the function and expression of OsVMT under Fe-excess conditions, it is likely to involve in Fe detoxification. Interestingly, the OsTOM3 DMA-efflux transporter gene belonging to Zinc-induced facilitator (ZIFL subfamily) was upregulated in all the tissues, thereby involved in metal transport. The characterization of the OsTOM3 gene needs to be further studied for better clarification about its role in Fe²⁺ toxicity. The OsPEZ1 induced in root tissue plays a significant role in the efficient translocation of protocatechuic acid (PCA) and caffeic acid from roots to shoots in rice (Ishimaru et al., 2011). Previously, it was found that expression of OsMIT increases under Fe excess, which is in accordance with the current study, essential for the proper growth and development of rice (Bashir et al., 2011). As reported earlier, OsYSL9 is involved in iron translocation, particularly from endosperm to embryo in developing seeds (Senoura et al., 2017). The YSL family transporters are responsible for transporting metal-phytosiderophores; however, their role is unclear in rice. OsYSL9 localizes in the plasma membrane and is believed to participate in the transportation of both iron (II)-nicotianamine and iron (III)-deoxymugineic acid into the cell. The expression of OsYSL10 is like OsYSL9 and may also participate in a similar mechanism. OsYSL13 was expressed in roots, and mature leaves participate in the distribution of Fe from older leaves to new leaves (Zhang et al., 2018). Earlier it was found that OsNRAMP2 was highly induced in shoots and participates in Cadmium accumulation (Zhao et al., 2018). Various studies have already identified the role of OsNRAMP2 to participate in Fe accumulation which is found to be upregulated in all the tissues. The OsARD1 is a metal-binding enzyme and is involved in the production of methionine as it binds with Fe²⁺ and catalyzes the formation of 2-keto-4-methylthiobutyrate (KMTB). In the current study, OsARD1 showed high upregulation in all the tissue types that might enhance rice tolerance under Fe²⁺ toxicity. Overexpression of OsARD1 also leads to the tolerance of abiotic stresses like submergence, drought, and salinity (Liang et al., 2019).

The discussion above suggests that the Fe exclusion and retention mechanisms in roots are likely to be insufficient under severe Fe²⁺ toxicity. ROS scavenging remains a key mechanism under severe Fe²⁺ toxicity which needs a better understanding (Aung and Masuda, 2020). In this study, most of the antioxidant genes were highly upregulated which clearly indicate the existence of ROS scavenging mechanism. Recently, few ROS scavenging genes, transcriptome factors and transporter genes were highlighted as probable candidates for the ROS scavenging system (Finatto et al., 2015; Wu et al., 2017; Aung et al., 2018; Aung and Masuda, 2020). However, specific ROS scavenging genes and their molecular mechanism are still unknown. The current study on RNA-Seq transcriptome explored the possible ROS scavenging system in rice under severe Fe²⁺ toxicity. Accordingly, a new defense mechanism (defense 4), i.e., ROS detoxification has been hypothesized as a possible mechanism of tolerance against severe Fe²⁺ toxicity. Excess Fe²⁺ is lethal to plants under acidic conditions as they catalyze H₂O₂ and produce a highly toxic hydroxyl free radical, termed as Fenton reaction (Winterbourn, 1995). Toxic hydroxyl free radical causes lipid peroxidation and promotes programmed cell death (Becana et al., 1998). Lipid peroxidation and programmed cell death can occur under different environmental stresses. However, Fe-mediated lipid peroxidation and programmed cell death results are quite different from others and are termed Ferroptosis (Distéfano et al., 2020). Rice has developed several Fe tolerance mechanisms, but only the ROS homeostasis was hypothesized to be essential and important in the tolerance mechanism in severe conditions (Aung et al., 2018; Aung and Masuda, 2020). Under Fe²⁺ toxicity, higher production of H₂O₂ was reported (Aung et al., 2018; Regon et al., 2021). H₂O₂ can be synthesized mostly in every cellular compartment, viz. peroxisomes, chloroplasts, mitochondria, nucleus, vacuoles, etc. CAT was reported to be mainly restricted only in peroxisomes with a very high concentration, thus removing H₂O₂ (Smirnoff and Arnaud, 2019). In concordance with previous reports, all three CAT genes were upregulated in roots and leaves of seedling and mature stages. Further, the involvement of these particular CATs was confirmed by KEGG analysis.

Most of the APX, ferredoxin, and thiol-based peroxidase such as PRX genes were upregulated in both roots and leaves, which are important for the removal of H₂O₂ in chloroplast using NADPH and photosynthetic electron transport via ferredoxin (2Fe-2S, iron-sulfur cluster binding domain-containing protein) as the ultimate reductant (Smirnoff and Arnaud, 2019). Several POX genes were upregulated, which are responsible for scavenging H₂O₂ by oxidizing various secondary metabolites. Removal of mitochondrial H₂O₂ requires a series of enzymatic detoxification processes. Mn-SOD scavenges superoxide radicals formed via mitochondrial electron transport chain (ETC). However, Mn-OsSOD1 was not found to be differentially expressed in roots, whereas it was fractionally upregulated in leaves of both stages. The remaining H₂O₂ is detoxified by the PRX-TRX system or by the enzymes of the ascorbate-glutathione cycle (Smirnoff and Arnaud, 2019). PRX-TRX plays an important role in detoxifying H₂O₂ in plants (Wang et al., 2016). The Os1-CysPrxB is highly expressed across all tissue types. In addition, upregulation of OsTRX24, OsTRX23, OsTRX2 etc. denotes the PRX-TRX regulation for detoxification of H₂O₂ in mitochondria under Fe²⁺ toxicity.

Among the different antioxidants, GSTs were one of the most differentially expressed genes under Fe²⁺ toxicity. Most upregulated GSTs are of the tau class, which are well-known for heavy metal detoxification due to the high affinity of metals to its thiol(-SH) group and as a precursor of phytochelatins (PCs) (Jagodzik et al., 2018). In this study, the tau GSTs such as OsGSTU6, OsGSTU8, OsGSTU17, OsGSTU19, OsGSTU30, OsGSTU44, etc., were highly upregulated in both root and leaves and thus expected to play an essential role in Fe²⁺ mediated cellular detoxification. The OsGSTU17 possesses diverse regulatory mechanisms in response to abiotic stresses, whereas overexpression of OsGSTU30 was reported to promote tolerance to Chromium (Cr) and drought stress (Yang et al., 2009; Srivastava et al., 2018). In addition, KEGG analysis revealed the glutathione pathways as one of the most actively regulated pathway in this study. Glutathione synthase viz. OsNADH-GOGAT1 and OsNADH-GOGAT2 were identified as highly upregulated genes in roots involved in the glutathione synthesis pathway, whereas OsNADH-GOGAT1 was identified in leaves. Moreover, alternative respiratory pathway component genes, like alternative oxidase (AOX) and NAD(P)H dehydrogenase (NDs), play an essential role in minimizing ROS production (Vanderauwera et al., 2005; Rhoads et al., 2006; Keunen et al., 2015; Huang et al., 2016). OsAOX1a, OsAOX1b, OsAOX1c, and OsNDB3 were highly upregulated in leaves, thus suggesting the existence of an alternative respiratory pathway under Fe²⁺ toxicity. As described previously, the modulation of this mechanism was observed under cold stress in rice (Ito et al., 1997). Overall, expression of GSH, APX, MDHAR, DHAR, and GR indicates the active role of the ascorbate-glutathione cycle under Fe²⁺ detoxification which has been also affirmed by the KEGG pathway analysis.

Iron homeostasis is a complex network that is regulated by various transcription factors. The tale of transcription factors involved in the regulation of different Fe-acquisition was well characterized in Arabidopsis, whereas it is comparatively less explored in rice. In Arabidopsis, 16 bHLH transcription factors were described to be involved in Fe deficiency responses (Gao et al., 2019). In this study, several other bHLH TFs were differentially expressed, which need to be further elucidated. Moreover, in Arabidopsis, MYB, WRKY, AP2/ERF, and C2H2 TFs were involved in Fe acquisition, translocation, inhibition, and modulation of other Fe homeostasis genes (Gao et al., 2019). In rice, OsIDEF1 (ABI3/VP1 TF family), OsIDEF2 (NAC TF family), and OsARF16 (ARF family) reported to modulate the expression of Fe-related genes and integrate auxin signals, respectively, thus play a critical role in Fe homeostasis (Ogo et al., 2008; Shen et al., 2015; Gao et al., 2019). Apart from the above TFs, several other TFs were differentially expressed under Fe²⁺ toxicity, which might involve Fe homeostasis regulation in rice.

Furthermore, TFs such as bHLH, bZIP, ERF, WRKY, NAC, and MYB were important for the protection of ROS mediated oxidative damage by triggering ROS scavenging related genes in plants (Mittler et al., 2004; Yu et al., 2017; He et al., 2018). However, the knowledge of TFs under Fe²⁺ toxicity is limited. Recently, NAC (OsNAC4, OsNAC5, OsNAC6) and WRKY TFs were hypothesized to regulate severe Fe²⁺ toxicity tolerance in rice (Aung and Masuda, 2020). Besides, GSNOR (downregulated in this study) was reported to promote root tolerance to Fe toxicity via a nitric oxide pathway (Li et al., 2019b). WRKY transcription factors are well-known to play a key role in both biotic and abiotic stress tolerance as well as plant hormones signal transduction and the MAPK signaling cascade in plants (Liu et al., 2007; Adachi et al., 2015; Kim et al., 2016; Jiang et al., 2017). Among the several upregulated WRKY TFs, OsWRKY8 and OsWRKY71 were involved in various biotic and abiotic stresses in rice (Liu et al., 2007; Kim et al., 2016). Moreover, APETALA2/Ethylene Response Factors (AP2/ERF) are well-known to regulate numerous abiotic stresses (Müller and Munné-Bosch, 2015; Phukan et al., 2017). Several AP2/ERF transcription factors were upregulated under Fe²⁺ toxicity. Interestingly, dehydration-responsive element-binding proteins (DREBs) transcription factors were downregulated in roots, whereas upregulated in leaves. Overall, differentially expressed TFs under Fe²⁺ toxicity are very intense, which need to be further studied to elucidate their specific role in understanding Fe²⁺ toxicity in rice. However, identification of their downstream target genes will remain important to uncover the possible signaling pathways in rice. Recent computational analysis has identified several cis-regulatory elements (CREs) and conserved motifs among the Fe homeostasis related genes which are important for their regulation by transcription factors (Kakei et al., 2021). Fe storage-related genes were reported to simultaneously possess the conserved downstream core element 1 (DCEp1) and Fe deficiency-associated motif 1 (FAM1) motifs, whereas Fe uptake genes tend to possess the FAM1, DCEp1, and TATABOX5 motifs (Kakei et al., 2021). Similar computational and molecular approaches of this study may be helpful in identifying novel mechanisms associated with Fe²⁺ toxicity tolerance in rice.

Comparative KEGG analysis revealed that genes involved in the Mitogen-activated protein kinases (MAPK) signaling pathway are highly upregulated in leaves, under Fe²⁺ toxicity. MAPK signaling pathway is a common defense response of plants for various abiotic stresses (Zhang and Klessig, 2001; Liu and He, 2017). Along with several MPK genes, OsMPK3 was upregulated in leaves, which are known as a stress tolerance gene (Jagodzik et al., 2018). In addition, pathogen-related protein 1 (OsPRI1) was also upregulated in all tissue types, which plays an essential role in the plant metabolism in response to biotic and abiotic stresses (Muhammad et al., 2019).

In addition, upregulation of low nutrient responsive genes indicates nutrient deficiency upon Fe²⁺ toxicity. Apart from the ROS homeostasis genes, differential expression of various abiotic stress-related genes might indicate the existence of multiple stress tolerance strategies in rice. Besides all the above, L-lactate dehydrogenase B (LDH-B) was the topmost upregulated gene in roots, which catalyzes the reversible NAD-dependent interconversion of pyruvate to L-lactate. KEGG pathway analysis revealed that it is involved in glycolysis/gluconeogenesis, cysteine and methionine metabolism, pyruvate metabolism, propanoate metabolism, and biosynthesis of secondary metabolites. Its stereoisomer D-2-hydroxyglutarate dehydrogenase/D-lactate dehydrogenase was recently characterized to be conferred multiple abiotic stresses by maintaining cellular homeostasis in rice (Mitsuhara et al., 2008). Thus, further study about LDH-B may be helpful to understand its function under Fe²⁺ toxicity in rice.

In this study, ROS homeostasis was identified as the key mechanism for Fe²⁺ detoxification under severe Fe²⁺ toxicity, regulated by transcription factors. Future molecular research work targeting the characterization of the strongly induced genes will be important to understand the overall Fe²⁺ detoxification mechanism in rice. In addition, the genes identified in this study may serve as a valuable insight for further research and development of rice genotypes for Fe²⁺ toxicity tolerance.

The original contributions presented in the study are publicly available. This data can be found here: Bioproject: PRJNA680441. BioSamples: SAMN16879912, SAMN16879913, SAMN16879914, SAMN16879915, SAMN16879916, and SAMN168799.

PR: conceptualization, methodology, writing–original draft preparation, data curation, and analysis. SD: manuscript writing and analysis. MR: methodology. AP: manuscript writing. UC: editing. BT: investigation, writing–reviewing, and editing. AT: reviewing and supervision. SP: reviewing, editing, and supervision. All authors contributed to the article and approved the submitted version.

The RNA Sequencing was funded by the Department of Biotechnology, Government of India with a grant no. DBT-NER/AGRI/29/2015.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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