The Effect of the Upregulated Expression of Fe Regulators IMA1 and bHLH104 in Arabidopsis on the Root Length and Fe Homeostasis Under Pi Starvation


 Phosphate (Pi) and iron (Fe) are two essential mineral nutrients for plant growth and development. Pi starvation triggers the Fe local redistribution and over-accumulation, resulting in the reduction of the primary root, while represses the expression of Fe uptake genes. Nevertheless, the antagonistic mechanism between P and Fe nutrition in plant remain not addressed. Here, the effect of the upregulated expression of Fe regulators IMA1 and bHLH104 driven by the different-type promoters (proCaMV 35S, the promoters of Pi-starvation responsive genes proIPS1 and proPHT1;4) in response to Pi starvation was investigated in Arabidopsis. The results showed that the expression of Fe uptake genes IRT1 and FRO2 was successfully upregulated in proIPS1::IMA1, proPHT1;4::IMA1 and proIPS1::bHLH104 under Pi starvation while decreased in pro35S::IMA1, pro35S::bHLH104 and proPHT1;4::bHLH104, compared with that in the corresponding plants under Pi sufficiency. Although the length and Fe distribution in roots of them didn’t have significant difference with wild type under Pi starvation, the Fe distribution and total Fe contents were significantly increased in shoots of proIPS1::IMA1, proPHT1;4::IMA1 and proIPS1::bHLH104 while were decreased in proPHT1;4::bHLH104. The higher Fe concentrations in the Pi-starved transgenic plants also conferred the obviously tolerance to Fe deficiency. Their biomasses and total P concentrations showed no difference with wild type, regardless of Pi sufficiency or deficiency. Therefore, this approach would be a novel manipulation to modify Fe nutrient via coupling with Pi starvation in plants.


Introduction
Phosphorus (P) is an essential mineral element for plant growth and development, involving in a myriad of important biological processes as a structural component in nucleic acids and phospholipids, in energy metabolism, in the regulation of enzymatic activities, and in signal transduction cascades (Hinsinger, 2001, Raghothama and Karthikeyan, 2005, Rausch and Bucher, 2002. However, most of soil P is rmly xed in minerals (e.g. Ca-P, Al-P and Fe-P) and cannot be directly used by plants, resulting in that the readily bioavailability (inorganic phosphate) represents a constraint for plant productivity in natural and agricultural ecosystems (Lynch, 1995). When suffering phosphate (Pi) starvation, plants modulate an array of morphological, biochemical and genetic modi cations, which include remodeling the root system architecture; enhancing the excretion of organic acids and phosphatases, nucleases and ribonucleases; activating Pi transporters to increase Pi uptake and utilization (Bates and Lynch, 2001, Chevalier, et Rouached, et al., 2010, Rufty, et al., 1993. Of these, the antagonistic interaction between Pi and Fe is thought as a proactive strategy in the remodi cation of the root architecture to cope with Pi shortage (Abel, 2017). Fe redistribution and accumulation as well as Fe-mediated ROS signals triggered by Pi starvation led to the inhibition of the primary root elongation, which could be rescued by decreasing ambient Fe status (Balzergue, et al., 2017, Huang, et al., 2018, Mora-Macias, et al., 2017, Ward, et al., 2008. Similarly, Fe plaques on rice root surface is obviously enriched under Pi starvation (Ding, et al., 2018). In addition, Hirsch et al. found that Pi de ciency promoted the modi cation of Fe storage from the vacuole to the chloroplasts (Hirsch, et al., 2006). Above all, alteration of Fe homeostasis plays crucial roles in adaptive response to Pi starvation.
At molecular level, the signaling pathways of Pi-starvation response are dissected into systemic and local responses depending upon the internal Pi status in plant and the external Pi availability, respectively (Gruber, et al., 2013, Lopez-Bucio, et al., 2002, Thibaud, et al., 2010. When Pi starvation is sensed, a large set of Pi-starvation responsive genes are induced to optimize Pi uptake and internal Pi utilization e ciency under the control of the master transcriptional factors PHR1 and PHR1-like 1 (PHL1) directly or indirectly (Bustos, et al., 2010, Puga, et al., 2017, Rubio, 2001). In addition, many Fe-de ciency responsive genes were also involved in the response to Pi starvation. The metal uptake and transport genes, such as IRT1, FRO2, NAS1 and NAS2 are downregulated in systemic responses to Pi de ciency while the repression could be relieved in mutant phr1phl1 (Bustos, et al., 2010, Thibaud, et al., 2010, indicating that the alteration of Fe homeostasis caused by Pi starvation is regulated by the central transcriptional factor PHR1. The expression of FERs, which are responsible for metal storage and detoxi cation, is locally induced under the control of PHR1 dependent on Pi homeostasis (Bournier, et al., 2013, Hirsch, et al., 2006, Ravet, et al., 2009, Thibaud, et al., 2010). An integrative analysis of multiple Pi-starvation and Fede ciency transcriptomic data exhibits that 579 different-expression genes are overlapped at both stresses. Of these, 90 genes are upregulated under Fe de ciency while downregulated under phosphate starvation in Arabidopsis root and 12 genes contain the P1BS cis-element in the promoter regions, which interacts with the transcription factor PHR1/PHL1 (Bustos, et al., 2010, Li andLan, 2015). Recent research showed that the core regulatory factor OsPHR1 and the possible Fe sensors OsHRZs form a reciprocal inhibition module to coordinate Pi and iron signaling and homeostasis in rice (Guo, et al., 2021). In brief, Fe-de ciency responsive genes is clearly pointed to be involved in the systemic response to Pi de ciency at transcriptional level.
In the local response of Pi starvation, Fe also plays crucial roles in the modi cation of root system architecture (RSA), such as inhibiting the elongation of primary root (PR), increasing the density and length of lateral root and root hairs, ultimately to increase the scavenging ability of Pi in soil, especially topsoil (Bates and Lynch, 1996, Gonzalez, et al., 2005, Gruber, et al., 2013, Ward, et al., 2008. In several long-root mutants (lpr1, lpr2, stop1, almt1), Fe accumulation in the root apoplast and cell wall is signi cantly less than that in wild type under Pi de ciency. On the contrary, the mutants (pdr2 and als3), whose roots are sensitive to Pi de ciency, always accumulate more Fe in the root ( Ticconi, 2004). More recently, the STOP1-ALMT1 pathway, which previously was responsible for the tolerance to low pH and Al toxicity, has been documented to be involved in Fe accumulation and redistribution, resulting in the reduction of primary root (Balzergue, et al., 2017, Hoekenga, et al., 2006, Luchi, et al., 2007, Mora-Macias, et al., 2017. The zinc nger-type transcription factor STOP1 activated the malate transporter gene ALMT1 to exudate malate, which contributes to Fe retention into root apoplast (Godon, et al., 2019, Hirsch, et al., 2006, Hoekenga, et al., 2006, Luchi, et al., 2007. The Fe accumulation triggers the peroxidase-dependent cell wall stiffening and Fe remobilization process across the different zones of the root (Balzergue, et al., 2017, Grillet, et al., 2014, Mora-Macias, et al., 2017. In addition, su cient Fe accumulation in nucleus promotes substantially accumulation of STOP1, which is repressed by another Al-toxicity responsive genes ASL3/STAR1 (Dong, et al., 2017, Godon, et al., 2019, Huang, et al., 2010, Larsen, et al., 2005. In root tip, the Fe-redox cycle mediated by the LPR1/2 initiates ROS, further contributing to Fe redistribution and callose deposition (Muller, et al., 2015, Svistoonoff, et al., 2007. The locally sequestration and redistribution of Fe 3+ in root tip induced the expression of CLE14 (Gutierrez-Alanis, et al., 2017). The secretion of peptide CLE4 is perceive by CLV2/PEPR2 to downregulate of the SHR/SCR and PIN/AUXIN pathway, which are essential for stem cell speci cation and maintenance in RAM (Benfey, et al., 1993, Bennett and Scheres, 2010, Di Laurenzio, et al., 1996, Meng and Feldman, 2010. Concomitantly, the interaction between LPR1 and PDR2, which encods a single P5-type ATPase, facilitates cell-speci c apoplastic Fe and callose deposition, further interfering the proper expression of SCR and the symplastic communication of SHR (Muller, et al., 2015). Taken together, Fe over-accumulation and redistribution in root tip as well as the Fe-induced oxidative stress in the apoplast of root tips cause the arrest of root cell elongation and the exhaustion of the root apical meristem, nally leading to the inhibition of the primary root. However, the root phenotype of mutant pdr2 under Pi starvation is independent on external Fe level (Ticconi, et al., 2009). Therefore, it remains to be explored that the alteration of Fe homeostasis causes the inhibition of the primary root triggered by Pi deprivation.
Considering the important roles of Fe nutrition in response to Pi starvation, therefore, we raised a hypothesis whether the enhancement of Fe uptake could relieve the inhibition of primary root triggered by Pi de ciency. In this work, we selected two functionally independent regulators IMA1 and bHLH104 in response to Fe de ciency and constructed the constitutive and inducible expression of them driven by different-type promoters of CaMV 35S as well as Pi-starvation responsive genes IPS1 and PHT1;4, respectively. The effect of the upregulated expression of Fe regulators on the expression of Fe uptake genes, root length and Fe homeostasis in response to Pi starvation was investigated. The study further developed the understanding of the antagonism between P and Fe. Moreover, this approach would be a novel manipulation to modify Fe nutrient via coupling with Pi nutrient in plants.

Plasmid construction and Plant Transformation
To construct the plant expression vectors, the sequence information of two promoters of P-de ciency responsive genes and two Fe-de ciency responsive genes was acquired from TAIR (The Arabidopsis Information Resource www.arabidopsis.org). The full-length cDNA of IMA1 (AT1G47400) and bHLH104 (AT4G14410) was ampli ed from the Fe-de ciency root RNA and cloned into SmaI and SalI sites between the CaMV 35S promoter and the NOS terminator of the binary vector pCAMBIA2301, resulting in the constructs pro35S::IMA1 cDNA and pro35S::bHLH104 cDNA, respectively. With respect to the inducible expression of IMA1 and bHLH104, the promoters of IPS1 (AT3G09922) and PHT1;4 (AT2G38940) were used to replace the CaMV 35S promoter in pro35S::IMA1 and pro35S::bHLH104 cDNA, respectively. The promoters of PHT1;4 and IPS1 were ampli ed in the genomic DNA that 2987bp and 1068bp upstream sequences from both genes' translation start sites, severally. All the primers used were listed in Table S1.
Then the modi ed binary vectors were transformed into Agrobacterium tumefaciens GV3101 to generate transgenic Arabidopsis. The T-DNA constructs were introduced into Col-0 plants via the oral dipping method as described previously (Clough and Bent, 1998).

RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufactures' instructions. 1 µg of the total RNA was used as the template to synthesize cDNA following manufactures' instructions of PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, with gDNA Eraser, Cat#RP047A). Afterwards, SYBR® Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, Cat# RR420A) was used for qRT-PCR on the Thermo PIKOREAL 96 Real-Time PCR System, with the following program: 95°C for 5 min, 95°C for 5 s, 60°C for 30 s, 40 cycles. The gene TUA3 (tubulin alpha-3, At5g19770) was employed as endogenous control to normalize the expression levels of target genes. The qRT-PCR primers used in this work were listed in Table S1.

Fe Histochemical staining assay
Fe( ) distribution in plants was observed using the Perls staining as the previous described method (Roschzttardtz, et al., 2009). In brief, the seedlings were vacuum in ltrated with Perls staining solution (containing 2% (v/v) HCl and 2% (w/v) K-ferrocyanide) for 15 min and incubated for another 30 min at room temperature. Then the seedlings were rinsed with ultrapure water for 3-4 times and imaged using a Zeiss Stemi 305 microscope.

Measurement of nutrient element contents
Shoots were harvested and washed with ultrapure water for 3-4 times. The samples were died for 2 days in a conventional oven at 70℃ after being blotted with tissue papers. The dry samples were digested completely in 65% ~ 68% HNO 3 at 120℃. The measurement of mineral (Fe, Mn and Zn) nutrients was determined by inductively coupled plasma spectroscopy (ICP-MS). The total P was measured based on the method of molybdate blue-colorimetric (Murphy and Riley, 1962).

Chlorophyll content analysis
The total chlorophyll content was measured according to the method described by Arnon (Arnon, 1949).
In brief, the shoots of Arabidopsis were collected and extracted in 80% acetone in dark until the pigments were decolorized completely. The absorbance values were recorded at the wavelength of 645 nm and 663 nm using a spectrophotometer (SpectraMax Plus 384 Absorbance Microplate Reader). Total chlorophyll content was calculated using the following formula: Chlorophyll content = (20.2 × A645 + 8.02 × A663)*V/FW. Hence, we raised a hypothesis that whether upregulating the expression of Fe-de ciency regulatory factors could reduce Fe accumulation on the root tip via enhancing Fe uptake, further relieving the inhibition of primary root triggered by Pi de ciency. Accordingly, we selected the upstream transcriptional factor bHLH104 in the Fe-de ciency regulatory network. And the inducible expression was constructed by combining with the promoters of Pi-starvation responsive genes PHT1;4 and IPS1, which play crucial roles in Pi absorption and sensing signal of Pi homeostasis in plants, respectively. Meanwhile, we also constructed the constitutive expression by CaMV 35S. The transgenic plants were generated and three independent lines for each transgene were determined by qRT-PCR ( Figure S1). Under Pi su ciency, the expression of bHLH104 was signi cantly higher in the transgenic plants of pro35S::bHLH104. Moreover, its expression was subtly upregulated in proPHT1;4::bHLH104 and proIPS1::bHLH104 compared with that in wild type. Correspondingly, the Fe uptake genes IRT1 and FRO2 were more highly expressed in pro35S::bHLH104. And the transcripts of IRT1 were slight higher in proIPS1::bHLH104 and proPHT1;4::bHLH104 in comparison with that in wild type whereas the expression of FRO2 was comparable (Table S2). The Fe regulator IMA1 was highly induced in pro35S::bHLH104 and proIPS1::bHLH104 while the expression of FIT was only higher in pro35S::bHLH104 than in wild type (Table S2). When 10-day-old seedlings were grown on Pi-deprived medium for 3 days, the expression of bHLH104 was more highly upregulated in transgenic plants than in wild type ( Figure S1 A-C). The expression of IRT1, FRO2 and IMA1 were higher in pro35S::bHLH104 and proIPS1::bHLH104 while showed no signi cantly in proPHT1;4::bHLH104 compared with that in wild type under Pi starvation (Table S2). In contrast, the expression of FIT was only higher in proIPS1::bHLH104 than that in wild type under Pi starvation (Table S2). Compared with that in the corresponding plants on the standard medium, in wild type, the expression of IMA1, IRT1 and FRO2 were inhibited under Pi starvation while the expression of FIT and bHLH104 showed little affected by Pi deprivation (Fig. 1A). In pro35S::bHLH104 and proPHT1;4::bHLH104, the expression of IRT1, FRO2 and IMA1 were inhibited under Pi starvation than that under Pi su ciency (Fig. 1A). However, the inducible expression of IRT1, FRO2 and FIT were successful in proIPS1::bHLH104 compared with that under Pi su ciency (Fig. 1A). Above all, the results indicated that the Fe-de ciency responsive genes were successfully induced by the upregulated expression of bHLH104 via the promoter of IPS1 while their expression was failed when bHLH104 was driven by the promoters of PHT1;4 under Pi starvation.
3.2 Upregulated expression of IMA1 driven by different-types promoters universally induced the expression of Fe uptake genes IRON MAN1 (IMA1), a novel member of small peptides harboring a short C-terminal amino-acid sequence consensus motif, is critical for the acquisition and cellular homeostasis of Fe as the potential hubs in the regulatory network of Fe-de ciency response (Grillet, et al., 2018, Kobayashi, 2019. Therefore, we sequentially constructed the constitutive and inducible expression of IMA1 to further explore the effect of Pi starvation on the expression of Fe uptake genes. Consistently, the expression of Fe uptake genes FIT, IRT1 and FRO2 was signi cantly higher in pro35S::IMA1 than in wild type, regardless of Pi supply, although their expression of them was decreased under Pi starvation compared with that in the corresponding plants under Pi su ciency ( Figure 1B, Table S2). In the transgenic plants of the inducible expression of IMA1, the expression of IRT1 and FRO2 was no signi cant difference under Pi su ciency while signi cantly upregulated under Pi starvation, compared with that in wild type. The expression of FIT was successfully induced in proPHT1;4::IMA1 while was slight decreased in proIPS1::IMA1 under Pi starvation in comparison with that in the corresponding plants under Pi su ciency ( Figure 1B). Combined with the results above, we found that the patterns of the expression of Fe uptake-associated genes were various in the inducible expression of IMA1 and bHLH104. As shown in Figure 1C, the expression of Fe uptake genes also showed signi cant difference among the transgenic plants of the upregulated expression of Fe regulators bHLH104 and IMA1. The Fe uptake genes were successfully induced in proIPS1::bHLH104 and proPHT1;4::IMA1 while their expression were inhibited in proPHT1;4:: bHLH104. Nevertheless, the expression of these genes also showed dramatically difference among these transgenic plants. Of these, the relative expression (-P treatment / +P treatment) of FIT and FRO2 was highest in proIPS1::bHLH104 while the IRT1 only showed the strongest induction in proPHT1;4::IMA1. In contrast, the expression of them was severely inhibited in proPHT1;4:: bHLH104. With respect to the different Fe regulators, the expression of FIT was more signi cantly induced in the transgenic plants of the upregulated expression of bHLH104 than that in the inducible expression of IMA1. On the contrary, the expression of IRT1 was more highly induced in proPHT1;4:: IMA1 and proIPS1::IMA1 than proIPS1::bHLH104. The expression of FRO2 was comparable in proPHT1;4:: IMA1 and proIPS1::bHLH104. The analysis of two-way ANOVA also revealed that the expression of FIT in proIPS1::bHLH104 showed signi cant higher than that in proPHT1;4::bHLH104 and proIPS1::IMA1 while no difference with that in proPHT1;4::IMA1. Nevertheless, its expression were comparable in the transgenic plants of the inducible expression of IMA1. In contrast, the expression of IRT1 displayed obvious dependence on the promoters. Its expression was signi cantly different between proPHT1;4::bHLH104 and proPHT1;4::IMA1 while the effect was little when they were induced under the control of proIPS1. The expression of FRO2 dramatically affected by the different promoters in the transgenic plants of the upregulated expression of bHLH104. Taken together, the expression of Fe uptake genes was differentially induced in the transgenic plants of the inducible expression of IMA1 and bHLH104 driven by the different promoters of Pistarvation responsive genes ( Figure 1D).

Effect of upregulated expression of IMA1 and bHLH104 on the root length and Fe distribution under Pi starvation
In view of the signi cantly differential expression of the genes associated with Fe uptake in transgenic plants under Pi starvation, therefore, we evaluated the effect of Pi starvation on the root elongation and Fe distribution in transgenic plants. When 7-day-old seedlings were grown on Pi-starvation medium for 7 days, the root length and biomass were signi cantly reduced compared with that on Pi-su ciency medium. However, the root length in transgenic plants showed no signi cant difference with that in wild type, regardless of Pi supply (Figure 2A

Upregulated expression of bHLH104 and IMA1 via Pi-starvation responsive promoters affects Fe distribution in shoots
To further investigate the effect of Pi deprivation on Fe homeostasis, we sequentially analyzed the Fe distribution in shoots of the upregulated expression of bHLH104 and IMA1. Under Pi su ciency, the results of histochemical staining of Fe ( ) displayed that visibly blue signals were observed in the overexpression of bHLH104 and IMA1 under Pi su ciency while not observed in the shoots of transgenic plant of the inducible expression as well as wild type. When grown on the Pi-de ciency medium, a few blue Fe signals were observed at the edge of shoots in wild type ( Figure 3A-D and S6 A-B). In contrast, the obviously more blue Fe plaques were aggregated in the shoots of pro35S::bHLH104, pro35S::IMA1, proIPS1::bHLH104, proPHT1;4::IMA1 and proIPS1::IMA1 than that in wild type while scarcely any blue Fe signals were observed in proPHT1;4::bHLH104 ( Figure 3A-D and S6 A-B). Nevertheless, the blue Fe plaques displayed signi cantly differences between the upregulated expression of bHLH104 and IMA1. For instance, in the shoots of transgenic plants related to bHLH104, the blue Fe plaques mainly enriched at the edge of leaves while the blue Fe signals were lled in the leaf vein of the whole shoots in the upregulated expression of IMA1 ( Figure 3A and C, S6 A). Therefore, we further measured the Fe contents in shoots (Fig. 3A-D and S6 C-D). Under Pi su ciency, the Fe contents were 24.9%~83.9% and 23.2% 8 2.2% greater in pro35S::bHLH104 and pro35S::IMA1 than that in wild type, respectively, while no signi cantly difference in transgenic plants of the inducible expression of them ( Figure S6 C-D). As shown in Fig. 3 and S6, Pi starvation signi cantly triggers the increase of the Fe contents in Arabidopsis. In comparison with that in wild type under Pi starvation, nevertheless, the Fe contents were increased by 20.2% ~ 58.4%, 118.0% ~ 134.3% and 45.7% ~ 106.1% in proIPS1::bHLH104, proPHT1;4::IMA1 and proIPS1::IMA1, respectively, while Fe content was decreased 16.0% ~ 45.1% in proPHT1;4::bHLH104 ( Figure 3E-H). Despite the different accumulation of Fe between them, the biomass of shoots showed no signi cant difference between transgenic plants and wild type whether under Pi su ciency or Pi de ciency (Figure S5 B, D and F). Considering the signi cant different Fe accumulation in transgenic plants under Pi starvation. Thus, we sequentially evaluated the chlorophyll contents when 5-day-old seedlings were grown on the medium with Pi (+P) or without Pi (-P) for 3 days and then they were transferred to the medium with Fe (+Fe) or without Fe (-Fe) for another 7 days (Fig. 4). Under +P+Fe and -P+Fe treatments, no signi cant difference was observed between transgenic plants and wild type. Under +P-Fe, in contrast, the chlorophyll content was 40.0% ~ 83.8%, 13.3% ~ 32.8%, 24.3% ~ 91.8% and 30.0% 52.9% greater in pro35S::bHLH104, pro35S::bHLH104, pro35S::IMA1 and proPHT1;4::IMA1, respectively, while showed no signi cant difference in proPHT1;4:: bHLH104 and proIPS1::IMA1, compared with that in wild type. When the Pi-starvation seedlings were transferred to Fe-de ciency medium, the chlorophyll content was 34.8% greater in wild type than that under +P-Fe conditions. Nevertheless, the chlorophyll contents in pro35S::bHLH104, pro35S::bHLH104, pro35S::IMA1 and proPHT1;4::IMA1 was still higher than that in wild type under -P-Fe conditions. The chlorophyll contents were subtly increased in proPHT1;4::IMA1 while showed reduced tendency in proPHT1;4::bHLH104 in comparison with that in wild type under -P-Fe conditions (Fig. 4). Nevertheless, the Pi contents were no signi cant difference between wild type and the transgenic plants ( Fig S7). Above all, the upregulated expression of IMA1 and bHLH104 driven by the promoters of Pi-starvation responsive genes PHT1;4 and IPS1 exhibited the different Fe distribution and Fe contents in shoots of Arabidopsis. The results also showed that these manipulation approaches would be novel to regulate the Fe nutrient in plants based on different purposes.

Effect of upregulated expression of IMA1 and bHLH104 on the contents of Zn and Mn
Given that Fe contents showed signi cant differences among the transgenic plants of the constitutive and inducible expression of IMA1 and bHLH104, we further assessed the effect on the content of other micronutrients, such as Mn and Zn. As shown in Table 1, the contents of Mn and Zn were signi cantly increased in the transgenic plants of the overexpression of IMA1 under Pi su ciency while there was no signi cant difference between the inducible expression of IMA1 and the wide type. Phosphate starvation triggered the Zn accumulation while decreased Mn content in wild type. Moreover, Zn contents were increased by 19.5% ~ 32.6% and 7.7% ~ 22.3% and 20.1% ~ 33.1% in pro35S::IMA1, proIPS1::IMA1 and proPHT1;4::IMA1 under Pi starvation, respectively. However, Zn contents were universally decreased in the transgenic plants of the upregulated expression of bHLH104, especially in proIPS1::bHLH104, where Zn contents were 18.5% ~24.9% less than wild type under Pi starvation. Mn contents showed the similar tendency with Fe contents in transgenic plants. The Mn contents were subtly increased in the transgenic plants in the upregulated expression of IMA1 while decreased in that of bHLH104. Above all, the upregulated expression of IMA1 obviously increased the content Zn and Mn while the upregulated expression of bHLH104 didn't. Combined with the Fe distribution and accumulation, therefore, the upregulated expression of IMA1 displayed more extensive function in regulation of micronutrients (Fe, Mn, Zn) while bHLH104 just had tissue-speci city expression in the regulation of Fe homeostasis.  . Moreover, the inhibition was alleviated by decreasing exogenous Fe concentration (Huang, et al., 2018, Ward, et al., 2008. In brief, Fe plays crucial roles in the systemic and local response to Pi starvation in plants. Hence, we wondered whether the upregulated expression of Fe uptake genes could reduce Fe accumulation on the root surface, further relieving the inhibition of primary root under Pi starvation. Sequentially, we constructed the inducible expression of bHLH104 and IMA1driven by the promoters of Pi starvation responsive genes IPS1 and PHT1;4 as well as the constitutive expression of them via the CaMV 35S promoter. Of these, bHLH104 is a key transcription factor in the regulatory network of Fe de ciency, while IMA1 is characterized as a long-distance interorgan signal involved in the uptake, translocation and cellular homeostasis of Fe in a wide range of plants. And both of them positively activated the expression of Fe-uptake system of FIT-FRO2/IRT1. (Grillet, et al., 2018, Kobayashi, 2019, Li, et al., 2016, Zhang, et al., 2015. In this work, we found that the expression of FRO2 and IRT1 was signi cantly inhibited under Pi starvation in wild type, consistent with previous reports (Fig S1).
Moreover, in pro35S::bHLH104 and pro35S::IMA1, their expression showed signi cantly decreased under Pi starvation than that under Pi su ciency although they were more highly expressed than that in the wild type, regardless of Pi supply ( Figure 1A-B, S1 C and F). In the transgenic plants of the inducible expression of Fe regulators, the expression of bHLH104 and IMA1 was successfully induced under Pi starvation, respectively ( Figure S1A-B, C-D). Nevertheless, Fe uptake genes IRT1 and FRO2 were differentially expressed in the transgenic plants. In proIPS1::bHLH104, the expression of FIT, FRO2 and IRT1 was highly induced under Pi starvation while failed in proPHT1;4::bHLH104 ( Figure 1A, Table S2), indicating that the regulatory expression of Fe uptake genes under the control of bHLH104 signi cantly depended on the promoters under Pi starvation. Previous research displayed that the primary expression patterns of bHLH104 were localized in the pericycle of the root maturation zone and veins of leaves maturation zone and in rosette leaves, cauline leaves, stems, and siliques but not in owers (Zhang, et al., 2015). The expression of IPS1 was mainly delimited by the endodermis of the root while PHT1;4, as a high a nity transporter, was predominantly induced in the epidermis, including the cortex and the root cap of root (Martin, et al., 2000, Misson, et al., 2004. Therefore, the higher induction of Fe uptake genes in proIPS1::bHLH104 seems to bene t from the functional overlap of IPS1 and bHLH104. However, the ectopic expression of bHLH104 might impair its precise function to control the expression of Fe uptake genes driven by proPHT1;4. Moreover, the results also sustained by recent research that the inducible expression of bHLH104 under control of MYB72 promoter enhanced the tolerance to Fe de ciency while the sensitivity to Fe de ciency was elevated when bHLH104 was driven by the promoter of IRT1 (Wang, et al., 2017). Similarly, MYB72 and IRT1 were strongly expressed in the root stele and in the root epidermis, respectively (Vert et al., 2002; Palmer et al., 2013). Therefore, the regulation of bHLH104 might exist tissue speci city in the Fe-de ciency regulatory network. In contrast, the expression of FRO2 and IRT1 was universally upregulated in the transgenic plants of the inducible expression of IMA1 ( Figure 1B, Table  S2), indicating that the upregulated expression of IMA1 displayed extensive regulation in Fe uptake. Rencent research also displayed that IMA1 have more broad effects functionally as the potential hubs in the regulatory network of Fe-de ciency response due to the extensive expression in companion cells and circulate in the sieve elements throughout the plant (Grillet, et al., 2018). Further investigation showed that the expression of Fe uptake genes were signi cant difference in the transgenic plants of the inducible expression of bHLH104 and IMA1 driven by different promoters (Figure 1C-D). IRT1 were more strongly expressed in the upregulated expression of IMA1 while FIT and FRO2 was more highly expressed in the transgenic plants of the upregulated expression of bHLH104, indicating that IMA1 might predominantly function in the direct regulation of Fe uptake while bHLH104 was mainly responsible for the regulation of FIT ( Figure 2C). Recently researches also illustrated that the IMAs genes, as a potential hubs of Fe-de ciency response, can directly activate bHLH39 independent on FIT while bHLH104 is able to regulate the expression of FIT by directly combining with the promoter of FIT (Hirayama, et al., 2018, Kobayashi, 2019, Zhang, et al., 2015. The two-way ANOVA also revealed that the IPS1 and PHT1;4 promoters displayed signi cantly distinct regulation to the expression of Fe uptake-associated genes in the transgenic plants of the inducible expression of IMA1 and bHLH104 ( Figure 2D).
Considering that the dramatically various expression of the genes responsible for Fe uptake in the inducible expression of IMA1 and bHLH104, therefore, we further evaluated the effect on the upregulated expression of IMA1 and bHLH104 in response to Pi starvation. Unfortunately, the root length as well as the biomass was no difference between wild type and transgenic plants, regardless of Pi-su ciency orstarvation conditions ( Figure 3C and D, Figure S2 E-H, Figure S4 and Figure S5). The Fe distribution and accumulation in roots depending on perls staining also showed no signi cant difference between wild type and transgenic plants although obviously blue Fe signals on the surface of roots were observed under Pi starvation compared with that under Pi su ciency ( Figure 3A and B, Figure Figure S5 A-B). However, no signi cant blue Fe signal was observed in the shoots of proPHT1;4::bHLH104 ( Figure 3A). Correspondingly, the Fe contents were decreased in proPHT1;4::bHLH104 while increased in proIPS1::bHLH104 compared with wild type, further sustaining that the ectopic expression of bHLH104 impaired Fe homeostasis in Arabidopsis ( Figure 3E and G). Unexpectedly, the Fe contents in shoot were greater in pro35S::bHLH104 under Pi su ciency while no signi cant difference under Pi su ciency compared with that in wild type ( Figure S5 C). In contrast, the Fe contents were signi cantly higher in the upregulated expression of IMA1 than in wild type and in the transgenic plants related to bHLH104 (Figure 4 ,S5). The results showed consistent with the expression of Fe uptake genes, suggesting that IMA1 and bHLH104 might had the different regulatory mechanism to the Fe-de ciency response ( Figure 1). Previous researches reported that the expression of bHLH104 was not affected by Fe de ciency while the activity of the transcription factor was controlled by BTS dependent on Fe homeostasis at post-transcriptional levels (Hindt, et al., 2017, Selote, et al., 2015. High Fe contents might contribute to the degradation of bHLH104. In contrast, IMA1 is positively induced in the early stage of Fe de ciency in companion cells and circulate in the sieve elements throughout the plant although the regulatory mechanism is still unclear (Grillet, et al., 2018).
Recently, a research reported that IMAs can compete with bHLH105 and bHLH115 for the interaction with the negative regulator BTS to elevate the Fe-de ciency response (Li, et al., 2021). Therefore, IMAs played more broad roles in Fe-de ciency regulation than bHLH104. Above all, the inducible expression of bHLH104 and IMA1 driven by different-type promoter signi cantly altered the Fe distribution and accumulation under Pi starvation. Furthermore, the chlorophyll concentrations also displayed the consistent tendency in them when Pi-starved seedlings were transferred to Fe-de ciency medium ( Figure  4). The above results indicated that the approaches were succeed in manipulating Fe homeostasis and the tolerance to Fe de ciency.
In order to improve Fe nutrient, lots of efforts have been done through upregulating expression of Feassociated genes. Masuda et al. induced the expression of the genes related to Fe storage protein ferritin (FERs) and Fe(II)-nicotianamine transporter (OsYSL2) via combining with endosperm-speci c promoters (OsGluB1, OsGlb1) or sucrose transporter promoter (OsSUT1), which signi cantly increased the Fe accumulation in rice seeds (Masuda, et al., 2013). However, it was less reported that the improvement of Fe nutrient by coupling the interaction between different nutrients. P immobility and high sorption capacity often results in the formation of Pi depletion zone in the rhizosphere (Lewis and Quirk, 1967). Moreover, the previous reports also indicated that overexpression of IMA1 and the inducible expression of bHLH104 via MYB72 promoter can dramatically increase the Fe content in seed of transgenic plants and the tolerance to Fe de ciency in alkaline soil (Grillet, et al., 2018, Wang, et al., 2017. In agroecosystem, Pi uctuation often happens due to the easy sorption and insolubility of P, resulting in the crops have to constantly forage Pi by the regulation of Pi uptake system. To acclimate to Pi de ciency, moreover, plants often increase the Fe retention on root surface while repress the expression of the genes responsible for Fe uptake (Ding, et al., 2018, Gutierrez-Alanis, et al., 2018, Hirsch, et al., 2006. Therefore, it would be an effective strategy to increase Fe nutrient by coupling with the regulation of Pi homeostasis. In this work, the inducible expression of IMA1 and bHLH104 signi cantly increased the Fe accumulation in shoots. In addition, the biomass and total P contents had no signi cant difference between wild type and transgenic plants under Pi starvation ( Figure S5 and S7). However, the expression of Fe uptakeassociated genes in root and the Fe contents in shoot were decreased signi cantly in proPHT1;4::bHLH104 ( Figure 1A, 3A and E). Altogether, induced expression of Fe-de ciency responsive genes can change the Fe distribution and accumulation via coupling with Pi starvation. Therefore, it also provided a potential strategy to manipulate Fe nutrient via the tissue-speci c expression of Fe-de ciency responsive genes.

Conclusion
The present study indicated that the upregulated expression of Fe-de ciency regulatory factors IMA1 and bHLH104 via the promoters of Pi-starvation responsive genes PHT1;4 and IPS1 successfully induced the expression of Fe uptake associated genes IRT1/FRO2 under Pi starvation, except for proPHT1;4::bHLH104, in which, they were repressed. The alteration of the expression of Fe uptake genes in Arabidopsis showed little effect on the root length and the Fe distribution in roots. However, the upregulated expression of Fe regulators obviously affected the Fe homeostasis in shoots of the transgenic plants. And the chlorophyll contents showed the similar tendency when Pi-starved seedlings were transferred to Fe-deprivation medium. The present study provided a novel strategy to manipulate Fe nutrient via speci cally induced Fe-de ciency regulatory factors. The authors declare that they have no con icts of interest.

Author Contributions
Ping Lan conceived and supervised the study; Xiangxiang Meng performed the experiments, analyzed the data and drafted the manuscript; Wenfeng Li and Renfang Shen provided infrastructure and polished the writing. The asterisks indicate the signi cant difference between the transgenic plants and wild type by Student's t-test (* p < 0.05, ** p < 0.01). The expression fold change (FC) were analyzed by two-way ANOVA analysis by Duncan's multiple range. (C) The data was showed mean ± SE calculated from 3 biological replicates.
(D) The different letters indicate the signi cantly difference among different transgenic plants (P < 0.05); an asterisk indicates there was a signi cant interaction between promoter and transgene in the effect on the expression of a gene (P < 0.05).

Figure 2
The effect of Pi starvation on the length and Fe ( ) distribution of roots in wild type and transgenic plants.
(A-B) The root length. (C-D) The perls staining in roots, bar = 1 mm. The root length and Perls staining were analyzed when 7-day-old seedling were grown on the Pi-starvation medium for 7 days. Bars in the graph showed mean ± SE calculated from 3 biological replicates. The asterisks indicate the signi cant difference between the transgenic plants and wild type by Student's t-test (* p < 0.05, ** p < 0.01). Figure 3