Molecular characterization of Fe-acquisition genes causing decreased Fe uptake and photosynthetic inefficiency in Fe-deficient sunflower

Iron (Fe) deficiency in plants hinders growth and yield. Thus, this study aims to elucidate the responses and molecular characterization of genes in Fe-deficient sunflower. The study was conducted on 14 days-old sunflower plants cultivated in hydroponic culture under Fe-sufficient and Fe-deficient conditions. The Fe-starved sunflower showed substantial decrease in plant biomass, SPAD score, quantum yield efficiency of PSII (Fv/Fm), photosynthetic performance index (Pi_ABS). Further, Fe shortage reduced Fe and Zn concentrations in roots and shoots, accompanied by a marked decrease of HaNramp1 and HaZIP1 expression in roots, suggesting the association of Zn status contributing to photosynthetic inefficiency in sunflower. The ferric chelate reductase (FCR) activity, along with HaFRO2 and HaIRT1 transcripts, were constitutively expressed, suggesting that sunflower plants can regulate FCR activity, although the lack of bioavailable Fe in the rhizosphere strongly corresponds to the limited Fe uptake in sunflower. The substantial increase of proton extrusion in roots and the localization of Fe-related genes in the plasma membrane are also evident in sunflower as common responses to Fe-deficiency by this Strategy I plant species. Analysis showed that three motifs of Fe-related proteins were linked to the ZIP zinc transporter. The interactome map revealed the close partnership of these Fe-related genes in addition to FRU gene encoding putative transcription factor linked to Fe uptake response. The cis-regulatory analysis of promoter suggested the involvement of auxin, salicylic acid, and methyl jasmonate-responsive elements in the regulatory process in response to Fe deficiency. These findings may be beneficial to develop Fe-efficient sunflower plants through breeding or genome editing approaches.


Morphological and photosynthesis features.
A digital caliper (Neiko 01407A Electronic Digital Caliper, United States) was used to measure the length of the roots and shoots. The dry weight of roots and shoots was taken after drying for 3 days at 80 °C in an electric oven. The leaf chlorophyll score was measured on a young leaf by a SPAD meter (Minolta, Japan). Furthermore, photosynthesis biophysics through chlorophyll fluorescence kinetic (OJIP), such as Fv/Fm (quantum efficiency of photosystem II), and Pi_ABS (photosynthesis performance index) were recorded on young leaves kept for 1 h in the dark using FP 100 PHOTON SYSTEM INSTRUMENT (CHECH REPUBLIC).

Analysis of Fe and Zn concentration in roots and shoots. Briefly, fresh roots were washed with
Milli-Q water and subsequently incubated at 4 °C in the first (10 mM MES) solution and in the second (10 mM MES + 1 mM EDTA), followed by washing 2-3 times in Milli-Q water. After surface cleaning, roots and shoots were kept on a falcon tube, keeping the lid open to dry at 70 °C for 3 days. Dried samples were subsequently digested with HNO 3 /HClO 4 (3:1 v/v) and made volume up to 10 ml. The solution was then used for elemental analysis based on standard curves by Atomic absorption spectroscopy (SHIMADZU, JAPAN).
Analysis of stress indicators. The standard curve for bovine serum albumin (BSA) was generated to estimate the total soluble protein, according to the Bradford assay 26 . Briefly, protein extraction was carried out by grinding the tissue samples with tris-HCL-buffer (50 mM, pH 7.5), 0.04% (v/v) β-mercaptoethanol, and 2 mM EDTA. The crude samples were centrifuged for 10 min at 12,000 rpm. The transparent fluid part was then mixed with 1 ml of Coomassie Brilliant Blue (CBB) before measuring the absorbance at 595 nm (60S UV-Visible Spectrophotometer, THERMO SCIENTIFIC, UNITED STATES). The concentration of unknown samples was then calculated based on the standard curve of different concentrations of BSA.
The electrolyte leakage demonstrating the loss of the cell membrane integrity were analyzed by a conductivity meter (HI98303, HANNA, UNITED STATES) 27,28 . Surface components of roots and shoots were dispensed frequently with deionized water. Thereafter, the freshly harvested samples were transferred into a beaker filled with 20 ml deionized water and kept at 25 °C for 2 h. Later, the solution's electrical conductivity (EC1) was calculated. Afterward, the samples were heated in a water bath for 20 min at 95 °C then soothed at 25 °C before recording the final electrical conductivity (EC2). The electrolyte leakage was then determined as follows: = (EC1/EC2) × 100 (%).
The cell death percentage was estimated using Evans blue (SIGMA-ALRICH, UNITED STATES) 29,30 . The freshly harvested root and shoot were washed with MilliQ water. The tissue samples were then incubated in 2 ml Evan's blue mixture for 15 min at room temperature. Afterward, 1 ml of the 80% ethanol was added to the mixture and incubated for 10 min at room temperature. The tubes with solutions were then incubated for 15 min at 50 °C in a water bath and then centrifuged for 10 min at 12,000 rpm. The absorbance of the supernatant was recorded at 600 nm. Finally, the percentage of cell death in the root or shoot tissue was evaluated based on sample fresh weight.
Fe chelate reductase activity. Fe (III)-FCR activity was determined in roots by ferrozine testing 5 . Briefly, the roots were washed once with 0.2 mM CaSO 4 and 2-3 times with Milli-Q water to eliminate the surface contaminants. The root samples were then homogenized with 1 ml of assay mixture (100 mM Fe(III) EDTA, 0.10 mmol MES-NaOH (pH 5.5), 300 mM ferrozine). The samples and blank tubes (without assay mixture) were incubated in the dark for 20 min at 25 °C. Finally, aliquots were read at 562 nm. The FCR activity was determined by using the ferrozine molar extinction coefficient (1.50 × 103 M −1 cm −1 ). Briefly, the pH of the cultivation medium was maintained by 0.1 M HCl or 0.1 M KOH by a digital pH meter. The H + efflux was measured by calculating the titrated quantity of acid or base to return pH to its starting point as follows: (amount of acid × concentration of acid) ÷ (fresh weight × time) 15 .
Estimation of total phenolics. The total phenolics concentration in roots was measured as previously described 29,30 . In short, the root extracts were mixed with 80% of Folin-Ciocalteu reagent and the solution of 20% of Na 2 CO 3 . The optical density of the solution was read at 765 nm. The unknown samples were calculated on the basis of the gallic acid calibration curve.
The qRT-PCR analysis. The total RNA was isolated from the fresh roots as described by SV total RNA isolation system (Promega, USA). The quantified RNA was then converted to cDNA using the cDNA synthesis kit (Promega, USA) before performing real-time PCR analysis in an Eco real-time PCR system (ILLUMINA, UNITED STATES) using gene-specific primers (Supplementary Table S1). The PCR reactions were set as follows: 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, 56 °C for 30 s. The relative expression of candidate genes was calculated considered Actin as an internal control by the dd −∆Ct method 31 .
Bioinformatics analysis. NCBI Blast program was run to retrieve the mRNA and protein sequences of HaIRT1, HaNramp1, HaZIP1, and HaFRO2. The CELLO (http://cello .life.nctu.edu.tw) server predicted the subcellular localization of proteins 27,28 . The MEGA (V. 6.0) developed the phylogenetic tree with the maximum likelihood (ML) method for 100 bootstraps using 11 HMA3 homologs from 11 plant species 32 . Furthermore, the ten conserved protein motifs of the proteins were characterized by MEME Suite 5.1.1 (http://meme-suite .org/ tools /meme) with default parameters, but five maximum numbers of motifs to find 33 . The interactome network of HMA3 protein was generated using the STRING server (http://strin g-db.org) visualized in Cytoscape 34 . The PlantCare was used for scanning of cis-elements present in promoter regions of these genes 35 .
Statistical analysis. At least three independent biological replications were considered for each sample in a randomized block design. The significance between +Fe and -Fe conditions was tested by t-test in Microsoft Excel 2007. In preparing graphic figures, GraphPad Prism 6 was used.
Ethical approval and permission. Formal ethical approval is not required for this experimental work as the sunflower line used in this work is a cultivated genotype. In addition, the seeds were collected from the local market; hence, permissions and/or licences for collection of seed specimens are not required complying with relevant institutional, national, and international guidelines and legislation.

Results
Plant growth, photosynthetic efficiency and elemental concentration. Along with the visual evidence, root and shoot morphological features (dry weight and length) significantly decreased owing to Fedeficiency in the hydroponic solution compared to Fe-sufficient sunflower plants ( Fig. 1a-e). In addition, the parameters associated with photosynthesis, such as SPAD score, Fv/Fm ratio, and Pi_ABS in young leaves, significantly declined due to Fe depletion relative to Fe-sufficient sunflower plants ( Fig. 1f-h). Results showed that Fe and Zn concentration significantly reduced in roots and shoots under Fe-deficiency compared to Fe-sufficient controls (Table 1).

Changes in stress indicators.
Fe-deficiency is known to induce stress in plants. In this study, the total soluble protein concentration in roots significantly reduced, although it remained unchanged in shoots due to Fe-deprivation compared to Fe-sufficient controls (Fig. 2a). Fe-depletion demonstrated a significant enhancement in electrolyte leakage and cell death (%) in both roots and shoots relative to controls (Fig. 2b,c).

Changes in strategy I responses in roots.
Fe-depletion caused no significant changes in root FCR activity in comparison with Fe-sufficient plants (Fig. 2d). In contrast, the proton extrusion activity in roots was significantly induced owing to Fe-shortage in comparison with Fe-sufficient plants (Fig. 2e). On the other hand, total phenolics secretion in roots remained unchanged between Fe-sufficient and Fe-deficiency controls (Fig. 2f).

Changes in the expression of Fe transporter and Strategy I genes. The expression of the HaIRT1
gene remained unchanged under Fe-depletion relative to Fe-sufficient controls (Fig. 3a). However, HaNramp1 and HaZIP1 were significantly downregulated in roots owing to Fe-deprivation in comparison with Fe-sufficient plants (Fig. 3a). However, the expression of HaFRO2 showed no substantial changes between Fe-sufficient and Fe-deficient conditions (Fig. 3a). The CELLO localization predictor showed that corresponding proteins of these genes are localized in the plasma membrane of roots (Fig. 3b). According to the phylogenetic tree, HaIRT1 and HaZIP1 are clustered together, while HaNramp1 and HaFRO2 are placed within-cluster, although all these genes are originated from the same ancestor (Fig. 3c).
In silico analysis of Fe-related genes/proteins. MEME motif analysis tool searched for the most conserved motifs identified in HaIRT1, HaNramp1, HaZIP1, and HaFRO2. Most of these ten motifs are located at site two and contain 6-50 residues (Fig. 4). Four out of ten motifs matched to particular domains are as follows: motif 1 (NPDNDJFFLIKAFAAGVILGTGFIHILPDAFDCLASKCLPEKPWGKFPF), motif 2 (HQFFEGIGLG-  (Fig. 4). Other motifs show no information (Fig. 4).
Interactome analysis was performed on the STRING server. The HaIRT1 shows functional partnership with FRO2 (ferric reduction oxidase 2) and FRU (a putative transcription factor) that regulates Fe uptake responses belonging to CL:28,152 local network cluster associated with siderophore-dependent Fe import into the cell (Fig. 5a). Functional enrichment of the network links to several biological functions, including cellular response to nitric acid, response to bronchodilator, Fe ion transport, cellular response to Fe ion, and Fe ion homeostasis. The HaNramp1 links to IRT1 (Fe-regulated transporter 1) and FRO2 (ferric reductase oxidase 2) partners under two local network clusters (siderophore-dependent Fe import into the cell and transition metal ion transmembrane transporter). The HaFRO2′s partners are IRT1 and FRU that regulated Fe uptake responses connected to CL:28,152 cluster (siderophore-dependent Fe import into the cell), having the same partner linked to the same biological processes observed for HaIRT1 (Fig. 5d). This gene cluster is related to several biological processes, which include Fe ion transmembrane transport, Fe ion transport, Fe ion homeostasis, divalent metal ion transport, and response to a bacterium (Fig. 5b). Further, the functional partners of HaZIP1 are RCK (ATP binding;  local cluster involved in chiasma assembly and meiosis protein (SPO22/ZIP4 like) linked to the chiasma assembly biological process (Fig. 5c).

Plant growth and photosynthesis under Fe deficiency. Although Fe-deficiency causes damages in
plants, a clear understanding of this nutritional stress in sunflower was hazy. In this study, Fe deficiency caused a severe reduction in length and biomass of roots and shoots in sunflower. In addition, the SPAD score dramatically dropped due to Fe starvation, suggesting that chlorophyll degradation in sunflower leaves are associated with the damages in photosynthetic apparatus due to Fe-deficiency. This was further supported by the decrease in the efficiency of photosystem II and photosynthesis performance index in Fe-starved leaves of sunflower. The reduction in the quantum yield of photosystem II is often associated with the Fe-deficient leaves 36 . Several studies also documented that a chlorotic leaf is closely correlated with photosystem II efficacy in plants under Fe deficiency 37,38 . A study demonstrated that the redox state of photosystem II acceptors was negatively affected due to Fe deficiency in sugar beet 39 . Moreover, proteins linked to the reaction center and light-harvesting antenna usually decline in Fe-starved leaves 40 . It implies that Fe-deficiency appears to inhibit the uptake of Fe but is also closely linked to photosynthetic inefficiency in sunflower. Furthermore, the changes in photosystem II parameters due to Fe-deficiency may be linked to damage in the reaction center or various elements of the energy transfer path in the photosystem II system in Fe-deficient sunflower plants. This message can be useful to strengthen the knowledge to avoid damage to the photosynthetic machinery in sunflower.

Changes in Fe concentration and transporter genes.
In this study, the HaIRT1 showed no changes in roots owing to Fe-deficiency, suggesting that this transporter is possibly not involved in Fe-deficiency tolerance in sunflower. In plants, a dual pattern of IRT1 expression was reported in Fe-depleted Arabidopsis 41 . Thus, it may be possible that the expression is highly dependent on the genotypic background of the cultivar/species and the duration of stress exposed to plants. In contradiction to the expression studies on Fe transporters, studies showed that the expression of IRT1 was induced in Fe-starved tomato 42 . Another study showed the role of Nramp1 in Fe mobilizing 42 , but it also cooperates with IRT1 to take up Fe in response to Fe-deficient conditions in Arabidopsis 43 . In this study, the HaNramp1 significantly downregulated in roots due to Fe-deprivation, suggesting that HaNramp1 is directly associated with the decreased Fe uptake along with its long-distance transport of Fe that eventually resulted in severe growth reduction and photosynthesis damages in sunflower. Also, ZIP proteins are involved in the uptake and transport of Fe and Zn in plants 14,16 . This is very much consistent with the decrease in Zn concentration and HaZIP1 expression in Fe-deficient sunflower roots. In general, the expression of ZIP genes is induced when plants get deficient in Zn, which facilitates cell Zn influx and Zn movement between organisms and also when plants become deficient in Fe or Mn 44,45 . Our results imply that HaZIP1 is also involved, at least in part, with Fe acquisition or fully dedicated to Zn uptake in sunflower plants or highly interacting with the Fe status of the sunflower plants. Furthermore, Fe-deficiency is also occurred due to excessive manganese in plants, inhibiting photosynthesis 46 . Consequently, photoinhibition of PSII may be the ultimate consequence of Mn exposure 47 . We, therefore, suggest that Fe-deficiency-induced reduction of Fe is tightly linked with the status of other essential elements, leading to the overall sensitivity to stress in sunflower plants. Moreover, the decreased Fe uptake and subsequent translocation largely attributed to the downregulation of several transporters (HaNramp1 and HaZIP1) involved in the uptake of Fe and Zn in roots, which agree with the severe chlorosis and photosynthesis damage in Fe-depleted sunflower. In this study, we assessed the status of Strategy I responses in Fedeprived sunflower plants. The FCR activity and its responsible HaFRO2 gene were consistently stable following Fe-starvation in the roots of sunflower. The constitutive expression or upregulation of the FRO1 in Fe-deficient plants, was reported in other plants 48,49 . However, the variations in FCR activity in response to Fe starvation were also reported in several Strategy I plants 15,50 . Studies also underscored the importance of FCR in Fe metabolism and photosynthetic efficiency in plants 51 . Not only that, the degradation of soluble proteins and the occurrence of the cell membrane damages in plants are often related to Fe-deficiency 5 , which was also evident in Fe depleted sunflower. Taken together, our findings confirm that the induction of FCR is a key factor in withstanding Fedeficiency in sunflower. Thus, the expression HaFRO1 is one of the strategies that can be modulated to improve Fe-efficiency in sunflower. Computational prediction suggests that HaIRT1, HaNramp1, HaZIP1, and HaFRO2 genes are localized in the plasma membrane. The Nramp and ZIP transporters are mainly distributed in the plasma membrane in mulberry 52 . Besides, IRT1 located in the plasma membrane has been established as one of the key plant model proteins for studying Fe acquisition and endomembrane trafficking 53 . We also constructed a phylogenetic tree in which HaIRT1 and HaZIP1 are clustered together while HaNramp1 and HaFRO2 are located in another cluster, but all these four genes are originated from the same evolutionary ancestor. The coordination of Fe and Zn transporters are widely known in Fe-deficient plants because of the similarities of these metal elements 54 .
Fe becomes unavailable to plants if the cultivation conditions have a pH higher than 8.0 15 . As a result, plants release H + in the rhizosphere to reduce the pH level 5,51 . This is a common Strategy I trait, enabling Fe acquisition that showed a substantial increase as evident from the proton extrusion activity in roots of sunflower. The proton extrusion involvement in Fe-depleted conditions was reported in other dicot plants 15,55 . Although the proton extrusion can contribute to Fe uptake of sunflower, this adaptive trait was a game-changer to maintain the overall Fe uptake system in Fe-deprived sunflower. Moreover, overcoming Fe-deficiency-induced is a complex process involving cumulative biochemical and molecular induction of Strategy I responses in sunflower plants. Further, the phenolic compounds released by roots responsible for Fe-chelating under Fe deficiency, as previously reported 56,57 , appear to be not involved with Fe homeostasis in sunflower plants.
In silico characterization of Fe-related genes. In recent years, in silico characterization of candidate genes before the wet-lab experiment is routinely performed to narrow down the target of studies. Conserved motifs are identical sequences of plant species that are maintained by natural selection. A highly conserved sequence has functional roles in plants and can be a useful start point for researching a particular topic of interest 58 . In this study, three motifs of HaIRT1 and HaZIP1 proteins are linked to ZIP zinc transporter. Several members of the Zn-regulated transporters and Fe-regulated transporter-like Protein (ZIP) gene family have shown to be involved in metal uptake and transport 59 . In maize, ZIP proteins are localized in cells playing important roles in the uptake of divalent ions 60 . Also, several ZIP genes are highly induced in roots and are involved in Zn uptake under Zn deficient conditions in barley 61 and bean 62 . In the light of these findings, it suggests that sunflower ZIP transporter might play a role in Fe and Zn uptake and distribution under low Fe availability.
The interaction network of a specific gene provides information about associations that may affect the regulations in response to particular stress in plants. The interactome map analyzed in the String platform showed a close partnership of FRO2 and FRU genes with sunflower with HaIRT1, HaNramp1, HaZIP1, and HaFRO2 genes generally linked to Fe uptake system in plants. Mutant studies showed that plants overexpressing FRO2 grew at a much higher rate than wild-type during Fe-deficient conditions 63 . Besides, the FRU gene is a mediator in inducing iron mobilization responses, indicating that iron uptake regulation is preserved in dicot species 64 . Thus, the biological functions of these sunflower Fe-related genes are involved in Fe ion transport and Fe ion homeostasis. Overall, this interactome finding might provide essential background for functional genomics studies of Fe uptake and transport in sunflower and related plant species.
Besides, promoter analysis reveals the involvement of cis-acting elements in HaIRT1, HaNramp1, HaZIP1, and HaFRO2 commonly associated with cis-acting elements in promoter and enhancer regions (CAAT-box) along with core promoter element around − 30 of transcription start (TATA-box). Other than HaNramp1, the Fe-related genes of sunflower are predominantly linked to cis-acting elements involved in the abscisic acid responsiveness, gibberellin-responsiveness, GATA-motif (part of a light-responsive element) and anaerobic induction. Abscisic acid is known to contribute to the response to oxidative damage in Arabidopsis thaliana 65 . In this study, the HaIRT1 promoter is linked to auxin and salicylic acid elements, while HaZIP1 and HaFRO1 promoters are generally attached to methyl jasmonate-responsive elements in sunflower. Auxin signaling affects Fe signaling and the Fe deficiency response in plants 66 . A recent study revealed that auxin plays systemic action on the expression of FRO1 in tomato 67 . Further, Fe deficiency induces salicylic acid signaling, thereby activating Fe translocation via the bHLH38/39-mediated transcriptional regulation of downstream Fe genes 68 . Gibberellin signaling controls the expression of transcription factor regulation iron-uptake machinery genes 69 , while jasmonate signaling is involved in the expression of Fe-deficiency induced genes in plants 70 . Studies demonstrated that promoter is anaerobically induced in plant tissues resulted in the synthesis of a new set of polypeptides or anaerobic stress proteins, but its connection with Fe-deficiency still needs to be established plants 71 . Altogether, our analysis implies that some hormones can be targeted to improve Fe-efficiency in sunflower.

Conclusion
This study gives new insights into the mechanical basis for Fe-deficiency responses in sunflower. Fe deprivation caused severe physiological and photosynthetic damage in sunflower. Further Fe-deficiency in sunflower resulted in a simultaneous decrease in Fe and Zn status of the plants. This physiological evidence was further supported by the downregulation of HaNramp1 and HaZIP1 transcripts in Fe-starved sunflower roots. Fe-related genes Scientific Reports | (2021) 11:5537 | https://doi.org/10.1038/s41598-021-85147-z www.nature.com/scientificreports/ (HaIRT1 and HaZIP1) are localized in the plasma membrane and are predominantly linked to motifs linked to ZIP zinc transporter. Interestingly, FRO2 and FRU partners showed a close association with HaIRT1, HaNramp1, HaZIP1, and HaFRO2 genes in sunflower in addition to the presence of cis-regulatory elements in promoters associated with auxin, salicylic acid, gibberellin, and methyl jasmonate-responsive elements. These results explore our understanding of Fe-starvation responses in the sunflower that can be further utilized in genome-editing or breeding programs to develop Fe-efficient genetic lines.