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 firmly fixed 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 modifications, 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 al., 2003, Li, et al., 2002, Lopez-Bucio, et al., 2002, Lynch, 2011, Lynch and Brown, 2001, Plaxton and Tran, 2011). Meanwhile, plant alters the homeostasis of other mineral nutrients to increase the acclimation of Pi-deficiency stress, which play crucial roles in regulating the Pi uptake and utilization and even affecting the growth and development (Baxter, 2009, Gniazdowska, et al., 1999, Hu and Chu, 2020, Linkohr, et al., 2002, Rouached, et al., 2010, Rufty, et al., 1993, Wang, et al., 2019). Of these, the antagonistic interaction between Pi and Fe is thought as a proactive strategy in the remodification 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, Gutierrez-Alanis, et al., 2017, Huang, et al., 2018, Mora-Macias, et al., 2017, Ward, et al., 2008, Zheng, et al., 2019). 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 deficiency promoted the modification 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 efficiency 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-deficiency 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 deficiency 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 detoxification, 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 Fe-deficiency transcriptomic data exhibits that 579 different-expression genes are overlapped at both stresses. Of these, 90 genes are upregulated under Fe deficiency 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 and Lan, 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-deficiency responsive genes is clearly pointed to be involved in the systemic response to Pi deficiency at transcriptional level.
In the local response of Pi starvation, Fe also plays crucial roles in the modification 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 significantly less than that in wild type under Pi deficiency. On the contrary, the mutants (pdr2 and als3), whose roots are sensitive to Pi deficiency, always accumulate more Fe in the root (Balzergue, et al., 2017, Dong, et al., 2017, Mora-Macias, et al., 2017, 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 finger-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, sufficient 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, Wang, et al., 2019). 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 Fe3+ 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 specification and maintenance in RAM (Benfey, et al., 1993, Bennett and Scheres, 2010, Di Laurenzio, et al., 1996, Gutierrez-Alanis, et al., 2017, Meng and Feldman, 2010). Concomitantly, the interaction between LPR1 and PDR2, which encods a single P5-type ATPase, facilitates cell-specific 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, finally 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 deficiency. In this work, we selected two functionally independent regulators IMA1 and bHLH104 in response to Fe deficiency 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.