A novel role of the calcium sensor CBL1 in response to phosphate deficiency in Arabidopsis thaliana

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Abstract

Phosphorus acts as an essential macroelement in plant growth and development. A lack of phosphate (Pi) in arable soil and phosphate fertilizer resources is a vital limiting factor in crop yields. Calcineurin B-like proteins (CBLs) act as one of the most important calcium sensors in plants; however, whether CBLs are involved in Pi deficiency signaling pathway remains largely elusive. In this study, we utilized a reverse genetic strategy to screen Arabidopsis thaliana T-DNA insertion mutants belonging to the CBL family under Pi deficiency conditions. The cbl1 mutant exhibited a relatively tolerant phenotype, with longer roots, lower anthocyanin content, and elevated Pi content under Pi deficiency, and a more sensitive phenotype to arsenate treatment compared with wild-type plants. Moreover, CBL1 was upregulated, and the mutation of CBL1 caused phosphate starvation-induced (PSIs) genes to be significantly induced under Pi deficiency. Histochemical staining demonstrated that the cbl1 mutant has decreased acid phosphatase activity and hydrogen peroxide concentrations under Pi deficiency. Collectively, our results have revealed a novel role of CBL1 in maintaining Pi homeostasis.

Introduction

Phosphorus (P) is an essential macronutrient for plant growth and development that plays vital roles in multiple biochemical processes in plant cells, including energy metabolism, signal transduction, photosynthesis, and respiration. Although P is abundant in soil, most organic and inorganic phosphorus are unavailable to plants. The concentration of phosphate (Pi), the inorganic form of P that is taken up and utilized by plants, is extremely low in soil (less than 10 μM) (Luan, 2009; Lambers et al., 2015). A lack of Pi leads to the accumulation of anthocyanin, with dark green leaves, growth retardation, and reduced yield. Thus, Pi deficiency is becoming one of the most limiting factors for sustainable crop production (Puga et al., 2017). In order to improve agricultural productivity, a large amount of Pi fertilizer is applied to soil, which causes serious environmental problems such as eutrophication of water sources (Conley and Likens, 2009). Hence, exploring mechanisms for achieving sustainable P utilization efficiency has become an urgent objective in plant breeding research (Heuer et al., 2017).

Pi deficiency seriously inhibits primary root growth and induces the formation of lateral roots and root hairs. To cope with Pi deficiency, plants have evolved a range of adaptations that improve Pi absorption and translocation, thus maintaining cellular Pi homeostasis (Rouached et al., 2010). The initial uptake and remobilization of Pi are regulated by Phosphate Transporter 1 (PHT1) proteins. PHT1 proteins play important roles in Pi uptake and allocation from the rhizosphere (Gu et al., 2016; Versaw and Garcia, 2017; Xu, 2018). PHT1;1 and PHT1;4, which are functionally redundant, mainly function in Pi uptake in roots under both low- and high-Pi conditions (Shin et al., 2004). PHO1, a member of the SPX-EXS subfamily, functions in Pi transfer from root epidermal and cortical cells to xylem and facilitates Pi translocation from root to shoot (Hamburger, 2002; Vogiatzaki et al., 2017). PHR1 (Phosphate-starvation Response 1) and PHR1-LIKE1 (PHL1), which have been identified as master transcription factors, play central roles in controlling the phosphate response of multiple targets by interacting with P1BS cis-elements, including PHTs, IPS1, RNS1, and SPX1 (Rubio et al., 2001; Bustos et al., 2010; Thibaud et al., 2010).

Calcium (Ca2+) is a ubiquitous second messenger in eukaryote cells that plays an important role in signal transduction in response to internal and external stimuli. Ca2+ signatures are perceived by Ca2+ sensors, such as calmodulin (CaMs), calmodulin-like protein (CMLs), calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs) families. The CBL protein harbors four elongation factor hands (EF-hands) which are responsible for Ca2+ binding (Luan, 2009; Hashimoto and Kudla, 2011; Kudla et al., 2018).

CBLs can form functional complexes with CBL interacting protein kinases (CIPKs), which not only respond to external adverse stimuli, such as drought, salt, cold, and immune stress responses, but also regulate the dynamic balance of diverse intracellular ions, including K+, Na+, Mg2+, NO3, and PO43- (Zhu, 2016; Kudla et al., 2018; Zhang et al., 2019; Chai et al., 2020; Lu et al., 2020). For example, the CBL1/9-CIPK23 complex activates AKT1 (Arabidopsis K+ transporter 1) and HAK5 (high-affinity K+ transporter 5) to enhance K+ absorption under low-potassium conditions (Xu et al., 2006; Ragel et al., 2015); moreover, the CBL1-CIPK23 complex can phosphorylate and activate CHL1/NRT1.1 (nitrate transporter1.1) to mediate both high-affinity and low-affinity nitrate uptake (Ho et al., 2009; Leran et al., 2015), and the CBL1/CBL9-CIPK23 complex can trigger the phosphorylation of S-type anion channel SLAC1 or SLAH3 to mediate stomatal opening (Tobias et al., 2014). The well-characterized salt overly sensitive (SOS) pathway comprising CBL4-CIPK24-SOS1 (a plasma membrane Na+/H+ antiporter) plays a vital role in salt tolerance (Liu et al., 2000; Qiu et al., 2002, 2004). CBL2/CBL3 with CIPK3/9/23/26 are involved in vacuolar Mg2+ sequestration to protect plants from Mg2+ toxicity (Ren-Jie et al., 2015). Additionally, previous studies have demonstrated that CBL1 mutations can impair plant responses to drought, salt, cold, glucose, and aluminum (Cheong, 2003; D’Angelo et al., 2006; Li et al., 2013; Ligaba-Osena et al., 2017). Although the CBL-CIPK complexes exhibit essential roles in the homeostasis of various ions in cells, whether they are also involved in the regulation of the Pi deficiency signaling pathway remains unknown.

In this study, we utilized a reverse genetic strategy to identify CBL1 as a negative regulator in the Pi deficiency signaling pathway in Arabidopsis. The isolated cbl1 mutant exhibited a relatively tolerant phenotype under Pi deficiency conditions and a more sensitive phenotype under arsenate treatment compared to wild-type plants. The CBL1 mutation led to increased transcription of multiple phosphate starvation-induced genes, including PHT1;1, PHT1;4, PHT1;5, IPS1, and RNS1. Moreover, the cbl1 mutant had decreased acid phosphatase activity and hydrogen peroxide concentrations. This novel discovery expands our understanding of CBL1 functions in Pi homeostasis.

Section snippets

Plant materials and growth conditions

Arabidopsis thaliana Ws seedlings (ecotype Wassilewskji), except for materials in Fig. S1, were used as the wild type in various experiments. The T-DNA insertion mutant lines including cbl1 (NASC ID: N9888), cbl2 (SALK_151426), cbl3 (SAIL_785_C10), cbl5 (GK-276F07), cbl7 (SAIL_100_F5), and cbl9 (SALK_142774) were obtained from the Nottingham Arabidopsis Stock Center (NASC). For phenotypic assays, seeds of Arabidopsis thaliana were surface sterilized with 8% NaClO (v/v) and then stratified for

The cbl1 mutant is tolerant to Pi deficiency and sensitive to arsenate

To determine whether CBL family proteins are involved in Pi deficiency signaling transduction, we implemented a reverse genetic strategy to screen genes encoding CBLs for T-DNA insertion mutants (cbl1, cbl2, cbl3, cbl5, cbl7, and cbl9) under Pi deficiency conditions (Fig. S1). Strikingly, we found that the cbl1 knockout mutant displayed a significantly tolerant phenotype under Pi deficiency conditions (LP, 50 μM), with much longer roots, fewer and shorter root hairs, and lower anthocyanin

Discussion

Pi is a macronutrient that is important for plant growth and development. However, the amount of available Pi in soil for plant growth is often limited, and about 70 % of global soil suffers from Pi deficiency, which has become a major factor limiting sustainable crop production (Chiou and Lin, 2011; Xu et al., 2019). Pi deficiency causes changes in plant root system architecture modifications, such as the inhibition of main root elongation and increased density and length of lateral roots and

Conclusion

CBL1 may function as a novel negative regulator of Pi homeostasis in plant cells under Pi deficiency, thus facilitating physiological adaptation of plants to constantly changing soils.

Author contributions

This research was designed by Wang C and Gao H. Gao H, Wang C, Li L, Fu D, Zhang Y, Yang P, and Zhang T performed the experiments. Gao H and Wang C analyzed the data. Gao H and Wang C wrote the manuscript with comments from all authors.

CRediT authorship contribution statement

Huiling Gao: Methodology, Investigation, Validation, Data curation. Chuanqing Wang: Conceptualization, Supervision, Writing - review & editing, Methodology, Investigation, Validation, Data curation. Lili Li: Methodology, Investigation, Validation, Data curation. Dali Fu: Methodology, Investigation, Validation, Data curation. Yanting Zhang: Writing - review & editing, Data curation. Peiyuan Yang: Writing - review & editing, Data curation. Tianqi Zhang: Writing - review & editing, Data curation.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This research was funded by a grant from Northwest A&F University (Z111021604 to CW), the National Natural Science Foundation of China (31770289 to CW), China Postdoctoral Science Foundation (2019M653756 to HLG), Natural Science Basic Research Plan in Shaanxi Province of China (2019JQ-210 to HLG and 2019JQ-135 to CW), the National Natural Science Foundation of China (31900218 to HLG), and partly supported by open funds of the State Key Laboratory of Plant Physiology and Biochemistry (

References (58)

  • S. Tu et al.

    Interactive effects of pH, arsenic and phosphorus on uptake of As and P and growth of the arsenic hyperaccumulator Pteris vittata L. Under hydroponic conditions

    Environ. Exp. Bot.

    (2003)
  • W.K. Versaw et al.

    Intracellular transport and compartmentation of phosphate in plants

    Curr. Opin. Plant Biol.

    (2017)
  • G. Xu

    Sensing and transport of nutrients in plants

    Semin. Cell Dev. Biol.

    (2018)
  • J. Xu et al.

    A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis

    Cell

    (2006)
  • J.K. Zhu

    Abiotic stress signaling and responses in plants

    Cell

    (2016)
  • C. Balzergue et al.

    Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation

    Nat. Commun.

    (2017)
  • R. Bari et al.

    PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants

    Plant Physiol.

    (2006)
  • R. Bhosale et al.

    A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate

    Nat. Commun.

    (2018)
  • R. Bustos et al.

    A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis

    PLoS Genet.

    (2010)
  • P. Catarecha et al.

    A mutant of the Arabidopsis phosphate transporter PHT1;1 displays enhanced arsenic accumulation

    Plant Cell

    (2007)
  • S. Chai et al.

    S‐acylation of CBL10/SCaBP8 by pat10 is crucial for its tonoplast association and function in salt tolerance

    J. Integr. Plant Biol.

    (2020)
  • Y.H. Cheong

    CBL1, a calcium sensor that differentially regulates salt, drought, and cold responses in Arabidopsis

    Plant Cell

    (2003)
  • T.J. Chiou et al.

    Signaling network in sensing phosphate availability in plants

    Annu. Rev. Plant Biol.

    (2011)
  • D.J. Conley et al.

    Ecology. Controlling eutrophication: nitrogen and phosphorus

    Science

    (2009)
  • C. D’Angelo et al.

    Alternative complex formation of the Ca2+-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis

    Plant J.

    (2006)
  • H.A. Del Vecchio et al.

    The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation

    Plant J.

    (2014)
  • B.K. Ham et al.

    Insights into plant phosphate sensing and signaling

    Curr. Opin. Biotechnol.

    (2017)
  • D. Hamburger

    Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem

    Plant Cell

    (2002)
  • S. Heuer et al.

    Improving phosphorus use efficiency: a complex trait with emerging opportunities

    Plant J.

    (2017)
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