Ferric-chelate reductase OsFRO1 involved in Fe reduction and long distance Fe transport channelized by OsYSL2 and OsYSL15 in rice

doi:10.21203/rs.3.rs-2242844/v1

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Ferric-chelate reductase OsFRO1 involved in Fe reduction and long distance Fe transport channelized by OsYSL2 and OsYSL15 in rice

https://doi.org/10.21203/rs.3.rs-2242844/v1

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Iron (Fe) is essential micronutrient that controls dynamic functions within cell of plants and its homeostasis is crucial for plant growth. In the present study, we found that a rice FRO gene, OsFRO1, dominantly expressed in young panicles and seedling. It is specifically responsible for Fe treatment and localized in the plasma membrane of cells. OsFRO1 positively interacts with two Fe transporters, Yellow Stripe-Like protein 2 (OsYSL2) and 15 (OsYSL15), which are further involved in long distance Fe transport. Knockdown of OsFRO1 (RNAi) suppressed the expression of the gene in Fe stress and substantially affected plant physiology and morphology with reduced Fe concentration in root and shoot of rice under Fe toxicity. Over-expression of OsFRO1 (OE) showed up-regulation and excess Fe excess increased ROS generation and plant senescence. In addition, the RNAi lines showed lower expression levels of OsYSL2 and OsYSL15 with less Fe toxicity and maintained greenness, modulated antioxidant activity compared to WT, whereas, the OE showed high transcript levels of the two gene and more Fe toxicity. All these results suggest that OsFRO1 functions in both Fe-reduction and Fe-uptake or translocation channelized by OsYSL2 and OsYSL15 transporters and plays crucial roles in Fe homeostasis in rice.

OsFRO1

Ferric chelate reductase oxidase

iron homeostasis

Fe-toxicity

rice

As the most crucial transition micronutrient, iron (Fe), was found existing in almost all living organisms, contributing to the redox centers of proteins for respiration, biosynthesis of chlorophyll, nitrogen fixation and photosynthesis (Suzuki et al., 2012; Vigani et al., 2013; Briat et al., 2015; Bashir et al., 2016; Li et al., 2019). The change in the oxidative state of Fe, Fe (II) to Fe (III), further stimulates cellular processes, electron transport, regulates metabolic functions and plant growth (Victoria et al., 2012; Suzuki et al., 2012; Vigani et al., 2013; Connorton et al., 2017). The activities of cellular organelles such as chloroplasts, plasma membrane and mitochondria are significantly correlated with Fe deficiency and toxicity, and the disturbance of Fe in these organelles by imbalance supply causes reduction in plant growth and development (Bashir et al., 2011a, b; Vigani et al., 2013b). Though, in chloroplast and mitochondria Fe functions as catalytic component, in the form of heme or Fe-S clusters, in a wide variety of proteins (Guerinot and Yi, 1994). Ferric reductase oxidase (FRO) is an intracellular enzyme that reduced ferric iron Fe (III) into ferrous iron Fe (II), playing important role in Fe homeostasis. Arabidopsis AtFRO7 (ferric reductase oxidase 7), a member of FRO family, is localized in chloroplast, functioning as reduction of Fe³⁺ to Fe²⁺ and subsequent uptakes Fe²⁺ into the cellular organelle (Jeong et al., 2008). AtFRO3 and AtFRO8 are positioned in mitochondria, suggesting the involvement of FROs in reduction of iron (Fe) at various developmental processes (Mukherjee et al., 2006; Jeong and Connolly, 2009). Loss-of-function of the rice mitochondrial transporter, MIT1, reduced Fe concentration in mitochondria, chlorophyll content, and also restricted plant growth (Bashir et al., 2011b; Mary et al., 2015). Vacuoles also serve as a largest reservoir of iron (Fe) storage, which can alleviate Fe toxicity (Moore et al., 2014). For example, rice vacuolar Fe transporter OsVIT1 and OsVIT2 are proposed to direct excess cytosolic Fe from tonoplast into the vacuole (Zhang et al., 2012). Likewise, the Arabidopsis vacuolar iron transporter-1 mutant (vit-1) is thought to accumulate Fe from the cytosol into the vacuole and responsible for Fe localization in seeds as well as deprived germination (Marschner, 1995). Although, Fe is found in abundance in soil, but the Fe bioavailability is restricted by two reasons: the high pH and calcareous soils (Alcantara et al., 2002). The major concern of low Fe is sparingly solubility and largely exists as insoluble hydroxides or oxides in alkaline soil, that is hardly available for plants (Mori, 1999).

The Fe deficiency in rice lowers productivity and produces stunted growth by reducing chlorophyll content and photosynthetic machinery, such as maximum quantum efficiency of PSII photochemistry (Fv/Fm), the potential photochemistry efficiency (Fv/F0), the electron transfer rate (ETR) and photochemical quenching of fluorescence (qP) (Stein et al., 2009; Sarvari et al., 2010). Additionally, Fe-deficient response can be seen in root architecture, like root hairs formation, root tip swelling, reduction in lateral root growth and low concentration of Fe in grain (Bouis and Welch, 2010; Bouis et al., 2011; Bouis and Saltzman, 2017). While Fe toxicity can cause damage in cellular component and rupturing, like DNA and proteins (Conte and Walker, 2011). For example, the water-logged anaerobic conditions with low soil pH (below 6.5), help in reduction of Fe³⁺ to Fe²⁺, which promotes the absorption rate of Fe, thereby causing cellular toxicity and ultimately loss of grain yield in rice (Santos and Oliveira, 2007; Stein et al., 2009a; Quinet et al., 2012). Furthermore, the excessive Fe in rice leaves exacerbates the generation of reactive oxygen species (ROS) in the redox centers, thereby causing the morphological changes, like typical symptoms leaf-bronzing and necrotic spots on leaves, which affect photosynthetic complex and finally reduce yield (10%-100%) (Audebert and Fofana, 2009). The hyper-accumulate free radicals in various tissues also participate in the Fenton reaction thereby generating cytotoxic hydroxyl radicals, and eventually oxidize chlorophyll and trigger degradation process. Thus, to avoid excess Fe, rice plant adopts three main strategies to alleviate excess ion-toxicity and maintain normal growth and development: (1) oxidation of Fe²⁺ to Fe³⁺ at root level (Deng et al., 2010; Connorton et al., 2017), (2) subcellular compartmentalization mechanisms or sequestering it in the state of ferritin (Briat et al., 2010a), and (3) ‘detoxification’ in the symplast (Majerus et al., 2007; Stein et al., 2009). Additionally, the regulatory mechanisms, signal transduction cascade and transcriptional levels are also triggered under iron excess condition in rice (Sperotto et al., 2010; Finatto et al., 2015). For the Fe uptake under Fe limiting condition, plant FROs acidify the rhizosphere to lower the soil pH, leading to the extrusion of protons H⁺-ATPases activity for Fe solubility and then the soluble Fe entered across the plasma membrane by transporters (Santi and Schmidt, 2009). This strategy is mainly used by non-grass species, while other is chelation-based strategy, where plants phytosiderophores (PS) are released into the rhizosphere (Kobayashi et al., 2010), and then bind to Fe (III) in soil, further Fe (III)-PS complexes are transported by different transporters family proteins (Curie et al., 2001; Inoue et al., 2009; Bashir et al., 2011; Ishimaru et al., 2011). The different strategies in graminaceous and non-graminaceous plants and molecular mechanism of Fe have been explained via diagrammatic sketch in supplementary Figure S2.

So far, studies have been mostly focused on Fe-deficiency in plants, while the regulation of Fe excess have remained largely unknown. Many genes encoding different proteins such as the Arabidopsis AtNAS1, AtNAS2, AtNAS3, and AtNAS4 (Suzuki et al., 1999), AtIRT1, AtIRT2, AtIRT3, AtZIP1 to AtZIP12 (Ishimaru, 2005; Ishimaru et al., 2006), rice OsNAS1, OsNAS2, and OsNAS3 (Victoria et al., 2012), OsIRT1, OsIRT2, OsZIP1 to OsZIP10 (Chen et al., 2008; Ogo et al., 2007; Lee et al., 2010), and two moss homologues PHY150995 and PHY215944 had showed greater copy number in response to low Fe-availability (Victoria et al., 2012). In addition, Arabidopsis AtNRAMP2, AtNRAM3, AtNRAMP4, AtNRAMP5 and rice OsNRAMP2 and OsNRAMP8 were found functioning in Fe storage, transport and subcellular compartmentalization under low availability of Fe (Mori, 1999; Curie et al., 2000; Bennetzen, 2002; Victoria et al., 2012). The Yellow Stripe-Like (YSL) gene family is also involved in metal transport (Koike et al., 2004; Inoue et al. 2009; Lee et al. 2009), for example, OsYSL2 is expressed in phloem and immature seeds under Fe deficiency (Koike et al., 2004). Furthermore, OsYSL15 is expressed in roots and critically involved in Fe uptake and homeostasis (Inoue et al., 2009). Maize yellow stripe1 (ZmYS1) is an orthologue of OsYSL2 and OsYSL15, which also has the ability to transport metal-nicotianamine (Curie et al. 2001; Aoyama et al. 2009). Arabidopsis AtYSL1, AtYSL2 and AtYSL3 (Waters et al., 2006) and TcYsl3 in T. caerulescens species, were also found participating in Fe absorption and transport by influencing nicotinamide (NA) complex (Mari et al., 2006; Stacey et al., 2008). Additionally, the iron-regulated transporter (IRT), transports Fe from root interface to upper portion of plant (Vert et al., 2002; Varotto et al., 2002). It was found that the IRT-1 and IRT-2 are mainly expressed in epidermal cells and responsible for Fe-translocation and balancing of ionic concentration in plant roots (Curie and Briat, 2003; Gross et al., 2003; Victoria et al., 2012). Moreover, study on Fe toxicity in rice seedlings have been approached through transcriptomic analysis (Quinet et al., 2012). Additionally, transcription factor OsWRKY80 is induced under Fe excess in plants (Ricachenevsky et al., 2010), but yet not fully functionally confirmed.

As mentioned above, FROs have ferric reductase activity and can reduce ferric iron Fe (III) into ferrous iron Fe (II). The plant FROs gene family have been reported in many species, including Arabidopsis (Wu et al., 2005), rice (Wang et al., 2013; Muhammad et al., 2018; Li et al., 2019), pea (Waters et al., 2002; Gross et al., 2003), and tomato (Li et al., 2004). Rice is the best model plant that utilizes two strategies for Fe uptake as described previously (Kobayashi et al., 2012). Rice genome contains only two FROs, these FROs have showed response under abiotic and metal stresses and hormone application (Muhammad et al., 2018), but their functional characterization and mechanism in Fe regulation is still not validated. In this study, we for the first time explored the role of OsFRO1 in Fe homeostasis and presented a new model that possibly utilizes for Fe regulation in rice plant. Moreover, the functional characterization of OsFRO1 under Fe toxicity in rice plant was also discussed in details.

Plant materials growth conditions and treatments

Rice (Oryza sativa L. japonica. CV. Nipponbare) wild type (WT) seeds were obtained from the State Key Laboratory of Crops Stress Biology for Arid Areas, Northwest A&F University, Yangling, China. Homozygous T3 transgenic lines of OE-OsFRO1 and RNAi-OsFRO1 were generated and were used for physiological experiments, tissue specific and phenology analysis. For gene expression analysis of transgenic plants, tissues, and leaf samples under metal stresses were set as described in our previous study (Muhammad et al., 2019), with a little modification of with or without of Fe supply in nutrient solution. Nutrient solution was regularly changed after 24 h and the plants were maintained in growth chamber at 16 h/8 h day/night, temperature cycle of 30^◦C/25^◦C, 800 µmol m^–2 s^–1light intensity and 60–65% relative humidity level. The Nicotiana benthamiana plants used for luciferase complementation imaging (LCI) assays and for subcellular localization analysis were grown separately in growth room by keeping 16 h 28^°C:8 h 22^°C, light: dark conditions and 3-week-old plants were used for the analysis.

Vector Constructs And Plant Transformation

For overexpression vector, the full-length open reading frame (ORF) of OsFRO1 gene (NCBI accession # AB126085, MSU LOC-Os04g48930), was amplified by PCR with high-fidelity Taq enzymes and specific primers listed in supplementary Table S1. The recovered PCR products were cloned into binary pCAMBIA-1301 vector under the control of Cauliflower mosaic virus (CaMV) 35S promoter and the OsFRO1-OE vector was obtained. For RNAi-interference vector, a partial CDS 251 bp sequence (bases 994–1245) of OsFRO1 gene with a set of specific primers (Supplementary Table S1) was cloned into the sense and antisense orientation of intermediate binary vector pTCK303 to get the interference OsFRO1-RNAi vector. For the expression pattern analysis of OsFRO1, the promoter region 1800 bp upstream of the ATG start codon of OsFRO1 was amplified and cloned into pCAMBIA 1301 binary vector and pOsFRO1:GUS vector was generated. During the construction of vectors, the standard method of molecular biology, protocol and reagents were used. The vector constructs of OsFRO1-OE, OsFRO1-RNAi and pOsFRO1 were introduced into rice cultivar (cv Nipponbare) via Agrobacterium mediated transformation method as described by Chen et al. (2003). Positive transformants were selected based on hygromycin and putative plantlets were confirmed by PCR. T3 homozygous lines were used for further experiments.

Subcellular Localization Of Osfro1 And Gus-histochemical Staining

Subcellular localization of OsFRO1 was investigated by transiently expressing GFP-OsFRO1 fusion into N. benthamiana epidermal cells and protoplast, respectively (Li et al., 2019). The full-length ORF of OsFRO1 was fused to green fluorescent protein (GFP) by KpnI and BamHI restriction enzymes, and the final construct (p35S:FRO1-GFP) was inserted into the pCAMBIA-1301 under the control of the constitutive CaMV 35S promoter, and then co-transformed into Agrobacterium tumefaciens strain GV3101 competent cells. The p35S-OsFRO1-GFP plasmid was transformed into N. benthamiana protoplast, thorough infiltration to 4- to 5 week-old the tobacco leaves at an OD600 (0.8–1.0) by Agrobacterium strain GV3101-mediated transformation, after incubation for 48h to 72h, the GFP signals were observed by an automated fluorescence microscope (OLYMPUS BX63, OLYMPUS, Tokyo, Japan). For the GUS histochemical staining, T2 seeds of pOsFRO1:GUS transgenic plants were germinated and grown in Hoagland’s nutrient solution for two weeks, and then the plants were transferred into plastic pots with filled field soils. Various tissues were collected and immersed in GUS staining solution (1 mM X-Gluc (5-bromo-4-chloro-3-indolyl-b-D-glucuronidase) overnight at 37 ^◦C. Stained samples were bleached with 70% ethanol for 24 h and were photographed by using a dissecting microscope (Leica, Germany).

Yeast Two-hybrid System

Yeast two-hybrid (Y2H) assay was performed by cloning the full-length CDS sequences of OsYSL2, OsYSL15 and OsFRO1 genes into pGADKT7 and pGBDKT7 vectors, respectively. The full-length cDNA of OsFRO1 was cloned into vector pGBKT7 as the bait vector, and the cDNAs of OsYSL2 or OsYSL15 were integrated into pGADT7 vector as the prey vectors. Subsequently, both the bait and prey plasmids were co-transformed into yeast strain Y2HGold according to the manufacturer’s instructions (Clontech, Dalian, China). The positive clones were ensured by growing into synthetic dextrose medium lacking of dropout media such as Leu, Trp, His, and Ade (LTHA) plus α-X-gal to determine the interactions.

Bimolecular Fluorescence Complementation (Bifc) Assay

This BiFC assay was performed as previously described (Zhang et al., 2020). In detail, the coding sequences of OsFRO1, OsYSL2 and OsYSL15 were amplified by PCR and cloned into pSPYNE and pSPVYCE vectors, respectively. Both cloning plasmids were co-transformed into Agrobacterium strain GV3101and then co-expressed in N. benthamiana leaves as earlier described (Hu et al., 2013). After 48 h infiltration, yellow fluorescence protein (YFP) signals were observed under a laser confocal microscope (Olympus FV1200, Japan) with 516 nm excitation and 532 nm emission wavelengths, respectively.

Firefly Luciferase Complementation Imaging (Lci) Assay

The LCI assays for the protein interaction detection was performed in tobacco leaves as described previously (Sun et al., 2013). The full-length CDS regions of OsFRO1, OsYSL2 and OsYSL15 were fused with the N- and C-terminal regions of the luciferase reporter gene LUC, respectively. Agrobacterium carrying the amino- and carboxy-terminal parts of LUC (nLuc and cLuc, respectively) derivative constructs were then infiltered into N. benthamiana leaves (Chen et al., 2008) and the LUC activity after 48 h infiltration were analyzed by NightSHADE LB 985 (Berthold, Bad Wildbad, Germany). Experiments were performed in three biological repeats for validity and similar results.

Rna Extraction And Gene Expression Analysis

Total RNA was extracted and cDNA were synthesized according to protocol described in our previous study (Muhammad et al., 2018; 2019). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed with Platinum SYBR Green qPCR Super Mix-UDG with ROX (Invitrogen, Carlsbad, CA, USA) on CFX96™ Real-Time PCR Detection System (BioRad, Foster City, CA, USA). OsActin1 (Gene ID: KC140126) was used as internal control and relative gene expression was calculated by the ΔΔ^Ct method. Primer sequences were listed in supplementary Table S1.

Analysis Of Metal Concentration, Germination Percentage And Photosynthesis Efficiency Under Fe Toxicity

The metal concentration was determined in plant roots, shoots, flag leaf and immature panicles. The samples were collected followed by drying for 3–4 days at 80 ^◦C in oven. Dried grinded powder weighing 0.1g was used for digestion. Metal concentration was analyzed by using inductively coupled plasma spectroscopy (ICP-MS, Agilent 7500ce, Santa Clara, CA, United States). Seeds (WT and RNAi) were grown on 1/2 MS medium with Fe-EDTA in different concentrations such as 50, 100, 200 and 300 µM, and the germination rates were measured after 24, 36, 48, 60, 72 and 84 h, respectively. The germination percentage was calculated by the formula: germination % = number of germinated seeds×100/total number of seeds. The experiment was repeated thrice for validation. The impact assessment of Fe toxicity in the photosynthetic efficiency was investigated on the first and second fully expanded leaves of transgenic rice. Greenness index (GI) was measured according to Huang et al., (2013). Quantum efficiency of photosystem II (PSII) was calculated as ΦPSII=(Fm’-Ft)/Fm’, where Fm’ is the maximal fluorescence and Ft is the steady-state terminal fluorescence. Other parameters, net photosynthesis rate and transpiration rate, were also monitored in standard methods by using a Li-6400 portable photosynthesis system (Li-COR Biosciences, Lincoln, NE, USA).

Detection Of Ros Production

In situ detection of H₂O₂ in leaves was conducted according to Wu et al. (2017). A. tumefaciens strain GV3010 harboring plasmids pCAMBIA1300-221-cMyc vector carrying the ORFs of OsFRO1, OsYSL2 and OsYSL15 or the empty vector were used to infiltrate 4-week old N. benthamiana leaves, respectively. In detail, the youngest and fully expanded leaf of N. benthaniana was detached and washed with 0.05% Triton X-100 followed by rinsing with distilled water several times. Then leaves were submerged into solution containing 0.5 mg mL^− 1 3,3^/-Diaminobenzidine tetrahydrochloride (pH 3.8) (Merck KGaA, Darmstadt, Germany) prepared in Milli-Q water (Merck KGaA) for 12 h in the dark, then leaves were rinsed with distilled water and subsequently bleached to remove chlorophyll with glycerol, lactic acid, and ethanol at a ratio of 1:1:4 (v/v), at 85 ^◦C for 1 h. ROS formation was visualized as browning precipitation by a scanner (CanoScan 9000F MarkII, Tokyo, Japan).

Statistical analysis

Statistical analysis (ANOVA) was performed using SPSS software (version 25.0, SPSS Inc., USA) and the mean values were compared by the Duncan test at the 95% confidence interval (p ≤ 0.05) for statistical significance. Experiments were run in three independent replications for each measured parameters, and the analyzed data were expressed as the mean values ± standard deviations (SDs). Analyzed data were plotted by GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).

OsFRO1 is differentially expressed in WT and transgenic plants of rice

Previously we found that a rice gene, OsFRO1 (LOC_Os04g36720), was substantially regulated by metal stresses (Muhammad et al., 2018). In order to elucidate its roles in Fe-sensing/homeostasis of rice, we cloned OsFRO1 from rice. As shown in Fig. 1A, OsFRO1 is expressed in all the tissues examined here with dominant transcripts found in the leaves at the tillering, booting and flowering stages in WT rice plants, suggesting high tissue specificity during plant growth and development of rice. The expression pattern of OsFRO1 was examined using the transgenic plants expressing the GUS reporter gene under the control of the OsFRO1 promoter. The GUS histochemical staining activity showed that OsFRO1 is significantly expressed in young seedlings and also panicles (Fig. 1D). To verify the specific role of OsFRO1 in Fe-regulation/involvement, we generated OsFRO1 over-expressed (OE) and OsFRO1 knock-down (RNAi) transgenic plant lines, via Agrobacterium-mediated transformation method (Supplementary Files Figure S1). The homozygous transgenic lines (OE and RNAi) were confirmed through PCR and three stable homozygous lines were established up-to T3 generation under natural conditions. Further, expression levels of OsFRO1 in three homozygous OsFRO1-OE and -RNAi transgenic lines were examined by qRT-PCR during seedling stage (leaf, root, stem) and reproductive stages (Pre-flowering stage, flowering stage and post-flowering stage), respectively (Fig. 1B), the results showed high expression levels in the tissues of OE lines, while the expression in RNAi lines was suppressed in all tested tissues. Likewise, the highest expression was recorded in the post-flowering panicles and seedling leaf blades of OE lines. Thereafter, the inducible expression levels of OsFRO1 under metals stresses were investigated by qRT-PCR. The results showed that the transcript of OsFRO1 was greatly stimulated by Fe treatment but not by other metals treatments in WT plants, while the mRNA abundance was reduced or even no detection by all metals stresses at each time point in RNAi lines (Fig. 1C), indicating that OsFRO1 is specifically responsible for Fe treatment. Further, the subcellular localization analysis in N. benthaniana epidermal cells showed that OsFRO1 protein is positioned at the plasma membranes (Fig. 1E), where further it was confirmed in the tobacco protoplasts (Fig. 1F).

OsFRO1 interacts with OsYSL2 and OsYSL15 and transient co-expression of OsFRO1 with OsYSL2 and OsYSL15 enhances ROS production

Previously, it was reported that OsYSL2 is critically involved in Fe translocation, especially in the shoots and endosperm (Ishimaru et al., 2010). Moreover, OsYSL2 localizes in the plasma membrane and involves in the transport of Fe(II)-NA and Mn(II)-NA complex through the phloem and load into grain. Likewise, the OsYSL15 is functioning in root Fe transport (Inoue et al. 2008; Lee et al. 2009). Whether or not the function of OsFRO1 in Fe homeostasis is related to OsYSL2 and OsYSL15, we then examined the interactive role of OsYSL2 and OsYSL15 with OsFRO1, initially by using STRING online tool (Supplementary files Figure S3). Further, we tested the interaction relationships of OsFRO1 with OsYSL2 and OsYSL15 by the firefly luciferase complementation imaging (LCI) assay. When the cLuc-OsYSL2 and cLuc-OsYSL15 were respectively co-expressed with nLuc-OsFRO1, strong fluorescent signals were observed in N. benthamiana leaves (Fig. 2A), indicating that OsYSL2 and OsYSL15 may interact with OsFRO1 in plants. Then, we practiced the bimolecular fluorescence complementation (BiFC) assay to test the interaction between OsFRO1 and the two YSL proteins, and strong interactions were also observed (Fig. 2B). To validate this result, the yeast two-hybrid system assay was employed to further examine the interaction relationship between OsFRO1 and OsYSL2 and OsYSL15 in yeast cells. Positive X-a-gal activity was observed in yeast containing pGBKT7-OsFRO1 plus pGADT7-OsYSL2 or pGADT7-OsYSL15, but not in the pGBKT7-OsFRO1 plus pGADT7-empty vector, indicating that OsYSL2 and OsYSL15 do interact with OsFRO1 (Fig. 2C). Based on these findings, it can be concluded that OsFRO1 has close interaction with OsYSL2 and OsYSL15 and they most possibly involved/participate in similar functions.

We further evaluated the co-expression analysis of OsFRO1with OsYSL2 or OsYSL15 by using a transient co-expression system. The results showed that transiently co-expression of OsFRO1 with OsYSL2 and/or OsYSL15 enhanced ROS production in N. benthamiana leaves (Fig. 2D and E). The deeper brown color of DAB staining was observed when OsFRO1 was respectively co-expressed with OsYSL2 or OsYSL15 as compared to the OsFRO1, OsYSL2 and OsYSL15 were expressed alone, indicating more polymeric oxidation products resulting from ROS accumulation were produced when the genes co-expressed. The transient co-expression of OsYSL2 and OsYSL15 also significantly increased the ROS production in the tobacco leaves (Fig. 2F). Together all, these observation suggests that OsFRO1 positively regulates ROS production, most probably by interaction with OsYSL2 and OsYSL15 for long distance Fe translocation.

Knock-down of OsFRO1 affects rice plant phenology and yield threshold ability

The knockdown of OsFRO1 transgenic plants (RNAi) exhibited a semi-dwarfism phenotypic effect and reduction in yield potential under field conditions. Although, OE plants were quite significant in regards to number of seed and size as well as other agronomic traits when compared to WT and RNAi plants (Table 1). Additionally, RNAi and WT rice plants displayed prominent phenotypic observation in several aspects (Fig. 3). Visual observations conformed that the reduced plant height was due to short internodes, culms and panicles size in RNAi plants (Fig. 3n). Other trait characteristics, the panicles of the transgene cover almost the normal height in later growth stages, but the distance between the secondary branches of panicle in rachilla were longer and attain less number in RNAi lines (Fig. 3b and d). There was no significant difference at the spikelet rachis at all developmental stages except the position of flag leaf in WT which is vertical along with spikelet and in RNAi plant that is projected horizontal or separated from spikelet, however at growing age (especially at heading stage) the panicle remained sheathed by the flag leaf (Fig. 3g and h).

Table 1

Comparison of agronomic traits of wild type (WT), knock-down (RNAi) and over-expressed (OE) plants.
Morphological traits	WT plants	OsFRO1-RNAi	OsFRO1-OE
Plant height (cm)	84.2 ± 2.1^b	80.4 ± 3.9^c	89.6 ± 3.2^a
Tiller number	9 ± 1.9 ^b	10 ± 2.3 ^ab	11.2 ± 1.3^a
Panicle length (cm)	23.2 ± 3.7 ^a	20.8 ± 1.5 ^c	21.2 ± 1.3 ^b
Flag-leaf width (cm)	1.40 ± 0.05^a	1.32 ± 0.11^c	1.34 ± 0.34^b
Flag-leaf length (cm)	26.3 ± 3.8^ab	24.7 ± 2.8^b	27.7 ± 1.4^a
Primary branch number	11.3 ± 2.3^b	13.7 ± 3.1^a	12.5 ± 1.2^ab
Secondary branch number	17.4 ± 1.6^b	17.8 ± 2.5^b	19.5 ± 2.3^a
Grain number per panicle	114.6 ± 7.9^b	103.0 ± 8.8^c	127.7 ± 3.2^a
Seed width (mm)	3.26 ± 0.10^a	2.95 ± 0.21^c	3.16 ± 0.03^b
Seed length (mm)	5.53 ± 0.33^b	5.50 ± 0.15^b	6.39 ± 0.35^a
Seed thickness (mm)	2.17 ± 0.19^a	1.98 ± 0.13^b	2.15 ± 0.12^a
1000-seed weight (g)	20.06 ± 0.13^b	16.82 ± 0.41^c	23.01 ± 0.58^a
Grain yield per plant (g)	13.34 ± 1.26^b	12.65 ± 1.48^b	16.19 ± 2.1^a

The base of flag leaf in RNAi transgenic lines were shrinking and curly structure with stunted growth of panicle as well as reduce grain size, while the base of WT was less rolled with longer leaf sheath leaf blade and normal panicle growth (Fig. 3i and j). In addition, the flag leaf showed Fe deficiency symptoms and brown spots with reduced chlorophyll in RNAi plants but not observed same symptoms in WT plants (Fig. 3k and l). Moreover, the flag leaf of RNAi plants showed browning from base and gradually increased at top (Fig. 3k and l). The maturity of RNAi panicles was also 15 days delayed from WT with significant reduction of yield in the number of seeds per panicle, with a subsequent effect of impaired empty florets, shrinked and reduced seed size. However, the OE grain showed globular and compact starch structure (Fig. 3C and D), but RNAi showed less compact, irregular shape (Fig. 3A and B), and disperse starch structure, when compared after cross-dissection of OE and RNAi grains (Fig. 3G and H). All these observations suggest that OsFRO1 regulation is probably associated with grain filling and knockdown of OsFRO1 loss efficiency of grain loading that resulted lower number of seeds, poor endosperm quality and nutrition deficiency, arrested plant growth, and limit final yield productivity of rice.

Knockdown of OsFRO1 affects plant growth and modulates rice plant physiology under normal Fe application

Since FROs function in Fe reduction in plants, we then checked whether knockdown of OsFRO1 affected the plant growth under normal Fe application. The WT and RNAi seeds were germinated and grown in MS-medium for further 10 days. The results indicated obvious differences in the roots of RNAi plants by comparing to WT (Fig. 4A). As the RNAi lines showed less roots bunches, roots hairs and reduced root length with poor root structure than WT roots (Fig. 4C, D and E). The root appearance and reduction in root characteristic may be due to the loss of function of OsFRO1 which retarded root growth and development of rice plants under normal condition. Further, the RNAi plants also showed considerable reduction in root activity (Fig. 4B), the root activity were reduced 50% after 4 days, while the WT showed less root activity reduction. In addition, we tested the germination performance of WT and RNAi transgenic lines under different Fe application conditions. The germinated seeds, which have expended plumule were counted and germination percentage was calculated (Fig. 4F). The results found that the function-less OsFRO1 in the knockdown RNAi lines markedly affected the germination percentage. We further noted that gradual increase with Fe apply reduced the germination percentage (Fig. 4F).

The antioxidant enzymatic activity protects plant from oxidative damage and balancing ROS-scavenging pathways as well as determines the ROS signaling network (Foyer et al., 2005). The antioxidant enzymes (SOD, POD, CAT, MDA) and proline contents were altered with the prolongation of time in both RNAi and WT plants (Supplementary Figure S4). However, the RNAi plants showed increase in the level of the SOD and APX activities and MDA content, while decrease in the level of the POD, CAT activities and proline content than the WT, suggesting that the knockdown of OsFRO1 significantly altered the antioxidant activity of the rice plants grown under Fe application and therefore possibly contributing to the ROS network and may help in adaptation strategy by modulating its antioxidant machinery. All these observations suggest the significant role of OsFRO1 in the plant healthy growth and thereby contributing in plant growth and development of rice.

Osfro1 Is Critically Involved In Long Distance Fe Transport Channelized By Two Metal Nicotianamine Transporters

To evaluate the Fe homeostasis in rice plant, we then checked the expression levels of the two metal-nicotianamine transporter genes, OsYSL2 and OsYSL15, in OsFRO1 transgenic OE and RNAi lines under Fe toxicity stress since the two transporters could interact with OsFRO1 at the molecular level. As depicted in Fig. 5, the RNAi lines have showed great sensitivity and substantially reduced the transcriptional abundance of OsYSL2 and OsYSL15 in both root and shoot, whereas the OE lines exhibited greater expression in root and shoot under Fe sufficiency as compared to WT (Fig. 5A and B). Noticeably, the OsYSL2 is markedly expressed in shoot and OsYSL15 is dominantly expressed in roots at later hours of Fe toxicity stress, respectively. Whereby, the Fe concentration in roots and shoots of the RNAi plants was obviously lower than that in WT under Fe sufficient condition, while, it was significantly higher in the roots following by shoots of OE plants compared with WT and peaked after 14 days, although the Fe concentration was decreased in all the different types of plants as the Fe stress was prolonged (Fig. 5C). The flag leaf is the source organ that remobilize various nutrients into panicle sink through vascular tissues during reproductive stage. In our results, the flag leaf showed much higher transcriptional abundance of OsFRO1 than that in immature panicle in OE than RNAi and WT plants (Fig. 5D), additionally, the Fe concentration in flag leaf was also much higher than immature panicle in OE by comparing to WT and RNAi plants (Fig. 5E). These results indicate that OsFRO1 efficiently functions in Fe uptake and/or translocation, most probably by channelized with OsYSL2 and OsYSL15. Therefore, it can be anticipated that Fe initially deposited in flag leaf at maturity stage and then simultaneously translocated into seeds during grain filling stage, where possibly the knockdown effect of OsFRO1 disturb Fe transportation from flag leaf to panicle sink. Upregulation of OsFRO1 influx greater amount of Fe content under Fe-sufficient conditions, which is further transported by OsYSL2 and OsYSL15 transporters into the other parts of plant, thus playing an important role in long distance Fe translocation in rice plant.

Overexpression of OsFRO1 leads to Fe overload, enhanced ROS accumulation and affected photosynthesis efficiency under Fe toxicity stress

To further understand the roles of OsFRO1 in Fe regulation, the two weeks old WT, OE and/or RNAi plants were treated with Fe stress (7 mM) for further two weeks and ROS activity and photosynthesis efficiency was measured. As shown in Fig. 6, the physical appearance of three OE transgenic lines depicted clear differences with bronze spots, necrotic lesion in all examined leaves, and more especially, the leaves near base of the stem have showed fast senescence compared with the RNAi transgenic lines (Fig. 6A). In addition, the survival rate of OE lines was greatly inhibited when excess of Fe was supplied to the culture solution for two weeks (Fig. 6B), while the RNAi lines sustained longer time and were remained greener as compared to WT plants. Histochemical stainings with DAB (3,3′-Diaminobenzidine) and NBT nitroblue tetrazolium were performed to evaluate the generation of ROS in RNAi and OE plants. The results indicated that the leaves of the OE transgenic plants showed much deeper color compared to RNAi leaves (Fig. 6C), implying that more ROS were accumulated in OE lines. Moreover, the generation of ROS was further verified by measuring the O₂⁻ and H₂O₂ production levels in both RNAi and OE transgenic lines (Fig. 6D and E). More O₂⁻ and H₂O₂ were generated in the plants under Fe stress, especially in OE plants. These results indicate that the overloaded Fe in cellular compartments increased ROS generation and Fe toxicity hindered cellular and enzymatic activity, that may trigger plant cell death (PCD).

In addition, we investigated the impact of Fe toxicity on the photosynthesis parameters in both OsFRO1-RNAi and -OE lines. The obtained results revealed that transpiration rate was significantly encountered by Fe treatment, and the OE plants showed lower transpiration rate under Fe stress as compared to RNAi lines (Fig. 6G). The OE plants also exhibited lower greenness index than the RNAi plants under Fe treatment (Fig. 6F). Regarding to net photosynthetic rate and photosynthesis efficiency, both RNAi and OE lines exhibited suppression under Fe stress, where the photosynthetic activity showed somewhat lower in OE plants than in RNAi plants with or without Fe stress (Fig. 6H and I). These results suggest that up-regulation of OsFRO1 accumulated high Fe density in OE leaves that appeared more Fe toxicity, while the RNAi plants due to loss-of-function of OsFRO1 have loaded less Fe in leaves and plants were remained healthy comparing to OE lines.

Previously, it has been reported that both Fe deficiency and overload negatively affect the plant growth and development. The Fe deficiency induces chlorosis and significantly affects photosynthetic components, thus leading to leaf and root senescence in rice plants (Stein et al., 2009), while the overloaded iron (Fe) in subcellular compartments induces damage at cellular level (Conte and Walker 2011). Therefore, plants have evolved a series of metal-ion detoxification mechanisms to control Fe uptake and homeostasis to maintain appropriate Fe concentrations (Guerinot and Yi, 1994). So far, a number of studies have reported that the role of various genes in the balanced regulation of Fe-uptake and homeostasis in plants. Here, we evaluated the OsFRO1 gene function in Fe homeostasis and morpho-physiological response under Fe stress in rice.

The Plasma Membrane Osfro1 Playing An Integral Role In Rice Growth And Development

Previous studies found that OsFRO1 significantly expressed in leaf/root, flag leaf and flowering stage (Muhammad et al., 2018, Wang et al., 2013). In Arabidopsis, AtFRO7 gene, a homologue of the homologue of OsFRO1 displayed high expression level in shoots, flower and silique (Wu et al., 2005; Jeong et al., 2008; Jeong and Guerinot, 2009; Hindt and Guerinot, 2012). Several reports found that AtFRO6 had high expression level in leave, root shoots and vasculature (Jain et al., 2014, Mukherjee et al., 2006). It was observed that FRO genes may play important role in the leaves roots and shoots under iron stress conditions in pea (Li et al., 2004, Waters et al., 2002). Recent studies found that the FRO exhibited high expression level in different photosynthetic tissues (Ishimaru et al., 2011; 2012; Kakei et al., 2012; Jain et al., 2014; Narayanan et al., 2015; Gama et al., 2017). Here, we found that the OsFRO1 gene was dominantly expressed in seedlings and flowering stages (Fig. 1A). The pOsFRO1-GUS stained transgenic tissues also showed high consistency to the expression data and markedly expressed in various seedling and spikelet’s (Fig. 1C). Based on these results, we postulated that FRO genes may play integral role in the growth and development, particularly in leaves and flowering related tissues in rice.

It was found that AtFRO1 protein is located in the chloroplast and reducing the ferric chelate reductase (FCR) activity in chloroplast (Wu et al., 2005; Jeong et al., 2008; Jeong and Guerinot, 2009; Hindt and Guerinot, 2012). The Arabidopsis AtFRO8 exhibited strong signals in the mitochondria of the cells (Jain et al., 2014). Previous study reported that OsFRO1 targets protein signal COOH-terminal domain to facilitate the trafficking of OsFRO1 protein to the plasma membrane (Jüschke et al. 2005). It has been reported that OsIRT1 and OsIRT2 are two other rice Fe-transporter proteins positioned in plasma membrane, functioning in Fe transport from roots to shoots (Bughio et al., 2002; Ishimaru et al., 2006). Here, we found that OsFRO1 displayed strong signals in the plasma membrane in the tobacco epidermal cells and protoplast (Fig. 1E). Thus, based on these evidences, we proposed that OsFRO1 may involve in Fe reduction (ferric into ferrous) and maintain Fe homeostasis by membrane associated activity in response to Fe supply or stress in rice.

Knockdown Of Osfro1 Significantly Affected Morphology And Yield Contributing Parameters In Transgenic Rice

Previously, it has been reported that the structure and size of grain significantly affect the final yield production. An improvement in grain shape and size through functional genomics effectively increase the quality of rice grain. Recently, the association of genes with quality traits have been identified and functionally characterized in rice plant (Luo et al., 2013; Si et al., 2016; Wang et al., 2020). It has been reported that GL3.1, GS2, and GW2 may play important role in the grain development in rice (Qi et al., 2012; Zhang et al., 2012, Song et al., 2007; Duan et al., 2016). A recent study found that GW8 of the SBP transcription factors family could promote the grain development in plants (Wang et al., 2012). In few decades, several genes have been identified which is associated with panicle morphogenesis and development such as Gn1a, LAX2, OsREL2, Hd1, DEP1, OsMADS1/17/29, WOX3 and PROG1 (Ashikari et al., 2005; Tan et al., 2008; Huang et al., 2009; Endo-Higashi and Izawa, 2011; Tabuchi et al., 2011; Yoshida et al., 2012; Nayar et al., 2013; Jiang et al., 2014; Hu et al., 2015; Wang et al., 2017). It has been reported that in graminaceous plants, the nutrients are translocated through phloem from the xylem and then transported to the flag leaf and spikelet regions (Yamaji and Ma, 2009). The functional characterization of osnac5 gene revealed on ABA-dependent abiotic stress-related regulator, where the highest expression was recorded in flag leaves of high iron and zinc rice cultivars (Sperotto et al. 2009; Takasaki et al. 2010; Song et al. 2011). Likewise, the GS1-RNAi transgenic plants functionally co-ordinates with reproductive organs and complete life cycle of rice (Le et al., 2018), and the TaNAM-RNAi transgenic wheat plants were accompanied with maintenance of protein and iron concentration in the flag leaf (Checovich et al., 2016). Here, we found obvious changes in RNAi plants by comparing to WT in various plant tissues, grain structure as well as reduction in final yield productivity (Fig. 3 and Table 1). The OsFRO1-RNAi plants displayed semi-dwarfism and less yield compared with the WT plants. We further noted that semi-dwarfism and less yield was due to short internodes, culms and panicles size in RNAi plants. Moreover, the RNAi plants displayed less number of panicle, the leaf flag was separated from spikelet, shrinking and curly structure with stunted growth of flag leaf, and reduce grain size compared with WT plants. We further noted that Fe deficiency and brown spots were found in the flag leaf of RNAi plants. The RNAi plants also exhibited late maturity and less number of seeds per panicle compared with WT plants. Taken together, the knockdown of OsFRO1 genes altered the growth and development of various parameters in rice, implying that OsFRO1 greatly contribute in rice plant morphology and contribute in grain structure, shape, and size.

OsFRO1 may promote the Fe homeostasis and channelize Fe (II) into various plant tissues by interacting with two transporter OsYSL2 and OsYSL15 genes

Previously, it was found that the proteasome-mediated turnover of bHLH is required for the uptake and homeostasis of iron (Zhang et al., 2020). Another study reported that MdBT1 and MdBT2 genes promote the iron homeostasis by targeting the bHLH in apple (Zhao et al., 2016; Zhang et al., 2020). The plasma membrane of the rice cells possessed Fe-transporter proteins (OsIRT1, and OsIRT2) that help in the Fe transport from roots to shoots (Bughio et al., 2002; Ishimaru et al., 2006). Additionally, the Fe plaque on root surface is considered as an essential factor affecting shoot Fe concentration in rice plants exposed to Fe toxic conditions, which depends on the root oxidizing power (Engel et al., 2012; Wu et al., 2014). The FRO1 promote the Fe homeostasis and the maintenance of high biomass when grown under Fe toxicity conditions in rice (Ruengphayak et al., 2015). Similarly, it was found that MuFRO1 mutant plants showed high tolerance in response to Fe stress compared with WT plants (Ruengphayak et al., 2015). Here, in this study, we found that the OsFRO1 gene displayed high expression level in response to Fe stress (Fig. 1C), and the greater accumulation of Fe concentration in roots and shoots of OE than WT and RNAi plants under Fe excess supply (Fig. 5C and E). We also noted that RNAi plants due to loss-of-function showed less ferric reductase activity in roots, as results less accumulation of Fe than WT plants and were remained healthier with high survival rate than OE plants under Fe toxicity stress (Fig. 6A and B), suggesting that OsFRO1 might involve in Fe uptake/translocation and upregulation under Fe toxicity stress.

It was found that TaNAM-RNAi plants maintained maximal photochemical efficiency modulated enzymatic network accompanied by low nitrogen and iron (Fe) concentration in flag leaf, suggesting that RNAi plants have more tolerance against Fe stress at certain level (Checovich et al., 2016). A quite similar pattern was also noted in the OsFRO1-RNAi plants. We found that RNAi plants showed highest photosynthetic efficiency than OE under Fe stress (Fig. 6F, G, H and I). Moreover, the RNAi plants modulated enzymatic activity but reduced the Fe concentration in roots followed shoots at seedling stage (Supplementary Figure S4, Fig. 5C). Based on these results, we concluded that less Fe accumulation due to knockdown effect of OsFRO1 maintained photosynthetic machinery from ion toxic damages and enhanced plant survival and performance under Fe stress.

The transporter genes associated with Fe uptake and translocation have been identified and over expressed under various promoters to facilitate iron (Fe) from root to shoot and ultimately in rice grains (Masuda et al., 2009; Lee et al., 2009b; Bashir et al., 2010; Kobayashi et al. 2012). The transgenic rice plants of seed-specific expression HvNAS1 by the glutelin promoter enhanced 1.5-fold higher amounts of Fe in the seeds (Usuda et al., 2009), and the over-expression by the rice Actin1 promoter also increased 2.3-fold higher level of Fe in polished grains (Masuda et al. 2009). Previous studies found that OsYSL2 (Koike et al., 2004), OsYSL15 (Inoue et al., 2009), OsIRT1 (Ishimaru et al., 2006) and OsFRDL1 may facilitate Fe (II) translocation to the shoot (Yokosho et al., 2009), suggesting that the regular Fe transporters play critical role in Fe homeostasis and transport. According to our hypothesis, the reduced Fe (II) can be transported from the membrane by Fe (II) transporters. We functionally confirmed the positive interaction of two metal-nicotianamine transporters, OsYSL2 and OsYSL15 with OsFRO1 proteins (Fig. 2). The yeast two-hybrid system, split LUC and BiFC assays found that OsFRO1 may play important role in the Fe homeostasis and long distance transport by targeting OsYSL2 and OsYSL15 in rice. We also noted that OsYSL2 and OsYSL15 genes displayed highest expression in OE than RNAi plants (Fig. 5A and B), further confirming the active e role of transporter genes in Fe translocation. Here, we further found that the high Fe accumulation in roots and shoot and flag leaf and immature seeds OsFRO1-OE plants, suggesting the individual role of OsFRO1 in Fe uptake and translocation. Based on these results, we concluded that OsFRO1 might plays dual functions as a Fe reduction and translocation into areal portion of plant, that require further verification.

The OsFRO1 belong to a super family of flavocytochromes located in the cellular membrane, functioning as Fe reduction and further translocate into aerial portion of plants. We found that Knockdown of OsFRO1 obstructed the process of Fe regulation as well as affected the plant physiology and morphology under normal Fe supply, while the OE plants accumulate higher Fe amount in root followed by shoot as well as significantly induced under Fe stress during seedling and reproductive stages. Moreover, the reduced Fe can be transported and subsequently loaded into phloem vessels by YSL transporters. The OsYSL2, a phloem located (Ishimaru et al., 2010) and OsYSL15, a xylem located (Inoue et al., 2009) transporters had showed significant expression levels under Fe stress in OsFRO1-OE plants by comparing to WT and RNAi transgenic rice lines, thereby proving biological significance of OsFRO1 in plant growth and development. Here, we are proposing the working model of OsFRO1 in chelate reductase activity and the interaction of two transporter genes involvement in long distance Fe transport in rice plant (Fig. 7).

Acknowledgements: We thankful to all the colleagues in our manuscript for providing useful discussions and technical assistance and providing constructive comments for its improvement.

Author contributions: I.M. conceptualized, conducted the experimental work, performed all the analysis and wrote the manuscript. Z.A and S.N helped in Data curation and experiments. J.X-Q, M.Z, A.S and L.T helped in the preparation of biological material and arranging laboratory materials. K.-M.C edited the manuscript and provide useful ideas. M.A.Z revised the manuscript. All the authors have read and approved the ﬁnal version of the manuscript.

Conflict of interest

The authors declare no competing financial interests.

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 31770204 and 31270299).

Ethics statement

The research has been conducted in laboratory and field and during research work no hazardous material were used which is harmful for any plant human or animal. The research is the original work and has not been plagiarized from any other sources.

Competing interests

The authors declare no competing financial interests.

Availability of data and materials

All datasets generated for this study are included in the article/Supplementary Material.

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Ferric-chelate reductase OsFRO1 involved in Fe reduction and long distance Fe transport channelized by OsYSL2 and OsYSL15 in rice

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Abstract

Figures

Introduction

Materials And Methods

Plant materials growth conditions and treatments

Vector Constructs And Plant Transformation

Subcellular Localization Of Osfro1 And Gus-histochemical Staining

Yeast Two-hybrid System

Bimolecular Fluorescence Complementation (Bifc) Assay

Firefly Luciferase Complementation Imaging (Lci) Assay

Rna Extraction And Gene Expression Analysis

Analysis Of Metal Concentration, Germination Percentage And Photosynthesis Efficiency Under Fe Toxicity

Detection Of Ros Production

Statistical analysis

Results

Osfro1 Is Critically Involved In Long Distance Fe Transport Channelized By Two Metal Nicotianamine Transporters

Discussion

The Plasma Membrane Osfro1 Playing An Integral Role In Rice Growth And Development

Knockdown Of Osfro1 Significantly Affected Morphology And Yield Contributing Parameters In Transgenic Rice

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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