A novel SlSTOP1-SlHAK5-cytosolic pH feedback loop drives dual adaptation to proton and aluminum toxicity in tomato

Abstract

Plants often experience aluminum (Al) toxicity in acidic soils, where the transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) plays a pivotal role in regulating transcriptional responses to Al stress. While posttranscriptional regulation of STOP1 under Al toxicity has been extensively studied, the mechanisms linking Al stress signals to STOP1 protein stability remain unclear. In this study, we employed multiscale pH imaging and noninvasive microtest (NMT) techniques to demonstrate that Al stress induces cytosolic acidification in the root apex of tomato (Solanum lycopersicum), which promotes the accumulation of SlSTOP1. This finding suggests that cytosolic acidification serves as a critical intermediate connecting Al stress to SlSTOP1 stabilization. Comparative transcriptomic analysis revealed that a significant subset of Al-responsive genes, including the known Al-resistance gene SlHAK5, are coregulated by both Al stress and low pH. Further functional characterization showed that SlHAK5 not only contributes to Al resistance but also plays a key role in maintaining cytosolic pH homeostasis under Al stress. In Slhak5 mutants, the expression of Al-induced genes was dysregulated, concomitant with attenuated cytosolic acidification. Correspondingly, SlSTOP1 accumulation was significantly reduced in Slhak5 mutants compared to wild-type (AC) plants under Al stress, indicating that SlSTOP1-mediated SlHAK5 expression feedback regulates cytosolic acidification. Additionally, Slhak5 mutants exhibited heightened sensitivity to proton stress. Collectively, our findings uncover a novel regulatory circuit involving SlSTOP1 and SlHAK5, which modulates SlSTOP1 stability through cytosolic acidification, thereby enhancing plant adaptation to proton and Al toxicity.

Acknowledgements

We are grateful to Dr Na Zhang (China Agricultural University) for providing us the antibody that against SlSTOP1, and Mr Longfei Zheng (Hefei Bapu Biotechnology Co., Ltd) for technical assistance in the H⁺ fluxes measurements. This work was financially supported by the Science and Technology Major Project of the Yunnan Province Science and Technology Department (no. 202502AE090025 to J.Y.), the Joint Agricultural Project of Department of Science and Technology of Yunnan Province (202401BD070001-010), the National Natural Science Foundation of China (No. 32372803), and Yunnan Province Ye Zhibiao Expert Workstation (202505AF350029).

Author contributions

Huihui Zhu and Yuzhi Bai (Conceptualization, Investigation, Methodology, Data curation, Writing—original draft), Liqiong Jia and Feifei Liang (Methodology, Data curation, Software, Investigation), Liqiong Jia, Chao Li, and Wei Fan (Data curation), Zheng-An Yang and Jianli Yang (Writing—review & editing, Funding acquisition)

Data availability

RNA-Seq data is available as accession number PRJNA1052400 for AC and Slhak5 mutants in response to Al stress treatment, and PRJNA1255724 for low pH stress of AC plants in the NCBI SRA database (https://www.ncbi.nlm.nih.gov).

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Delhaize E, Ryan PR. Aluminum toxicity and tolerance in plants. Plant Physiol. 1995; 107:315-21

[2] Ma JF. Syndrome of aluminum toxicity and diversity of alu-minum resistance in higher plants. Int Rev Cytol. 2007; 264: 225-52

[3] Zheng SJ, Yang JL. Target sites of aluminum phytotoxicity. Biol Plant. 2005; 49:321-31

[4] Kochian LV, Pineros MA, Liu J. et al. Plant adaptation to acid soils: the molecular basis for crop aluminum resistance. Annu Rev Plant Biol. 2015; 66:571-98

[5] Ma JF, Ryan PR, Delhaize E. Aluminium tolerance in plants and the complexing role of organic acids. Trend Plant Sci. 2001; 6:273-8

[6] Kochian LV, Hoekenga OA, Pineros MA. How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Annu Rev Plant Biol. 2004; 55:459-93

[7] Yang JL, Fan W, Zheng SJ. Mechanisms and regulation of aluminum-induced secretion of organic acid anions from plant roots. J Zhejiang Univ-Sci B. 2019; 20:513-27

[8] Sasaki T, Yamamoto Y, Ezaki B. et al. A wheat gene encoding an aluminum-activated malate transporter. Plant J. 2004; 37:645-53

[9] Furukawa J, Yamaji N, Wang H. et al. An aluminum-activated citrate transporter in barley. Plant Cell Physiol. 2007; 48:1081-91

[10] Magalhaes JV, Liu J, Guimaraes CT. et al. A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nat Genet. 2007; 39:1156-61

[11] Iuchi S, Koyama H, Iuchi A. et al. Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance. Proc Natl Acad Sci USA. 2007; 104: 9900-5

[12] Sawaki Y, Iuchi S, Kobayashi Y. et al. STOP1 regulates multiple genes that protect Arabidopsis from proton and aluminum tox-icities. Plant Physiol. 2009; 150:281-94

[13] Zhang Y, Zhang J, Guo JL. et al. F-box protein RAE1 regulates the stability of the aluminum-resistance transcription factor STOP1 in Arabidopsis. Proc Natl Acad Sci USA. 2019b; 116:319-27

[14] Cao HR, Zhang M, Zhu X. et al. The RAE1-STOP1-GL2-RHD6 module regulates the ALMT1-dependent aluminum resistance in Arabidopsis. Nat Commun. 2024; 15:6294

[15] Fang Q, Zhang J, Zhang Y. et al. Regulation of aluminum resis-tance in Arabidopsis involves the SUMOylation of the zinc finger transcription factor STOP1. Plant Cell. 2020; 32:3921-38

[16] Xu JM, Zhu JY, Liu JJ. et al. SIZ1 negatively regulates aluminum resistance by mediating the STOP1-ALMT1 pathway in Ara-bidopsis. J Integr Plant Biol. 2021; 63:1147-60

[17] Zhou F, Singh S, Zhang J. et al. The MEKK1-MKK1/2-MPK4 cas-cade phosphorylates and stabilizes STOP1 to confer aluminum resistance in Arabidopsis. Mol Plant. 2022; 16:337-53

[18] Ding ZJ, Xu C, Yan JY. et al. The LRR receptor-like kinase ALR1 is a plant aluminum ion sensor. Cell Res. 2024; 34:281-94

[19] Wei X, Zhu Y, Xie W. et al. H2O2 negatively regulates aluminum resistance via oxidation and degradation of the transcription factor STOP1. Plant Cell. 2024; 36:688-708

[20] Tokizawa M, Enomoto T, Ito H. et al. High affinity promoter bind-ing of STOP1 is essential for early expression of novel aluminum-induced resistance genes GDH1 and GDH2 in Arabidopsis. JExp Bot. 2021; 72:2769-89

[21] Tian WH, Cai WY, Zhu CQ. et al. STOP 1 regulates CCX1-mediated Ca2+ homeostasis for plant adaptation to Ca2+ deprivation. J Integr Plant Biol. 2024; 66:2126-39

[22] Godon C, Mercier C, Wang X. et al. Under phosphate starvation conditions, Fe and Al trigger accumulation of the transcription factor STOP1 in the nucleus of Arabidopsis root cells. Plant J. 2019; 99:937-49

[23] Tian WH, Ye JY, Cui MQ. et al. A transcription factor STOP1-centered pathway coordinates ammonium and phosphate acquisition in Arabidopsis. Mol Plant. 2021; 14:1554-68

[24] Zhu H, Chen W, Za Y. et al. SlSTOP1-regulated SlHAK5 expression confers Al tolerance in tomato by facilitating citrate secretion from roots. Hortic Res. 2024;11:uhae282

[25] Le Poder L, Mercier C, Fevrier L. et al. Uncoupling aluminum toxicity from aluminum signals in the STOP 1 pathway. Front Plant Sci. 2022; 13:785791

[26] Gong XQ, Shi ST, Dou FF. et al. Exogenous melatonin allevi-ates alkaline stress in Malus hupehensis Rehd. by regulating the biosynthesis of polyamines. Molecules. 2017; 22:1542

[27] Zhang F, Yan XY, Han XB. et al. A defective vacuolar proton pump enhances aluminum tolerance by reducing vacuole sequestra-tion of organic acids. Plant Physiol. 2019a; 181:743-61

[28] Ozkan P, Mutharasan R. A rapid method for measuring intracel-lular pH using BCECF-AM. BBA-Gen Subjects. 2002; 1572:143-8

[29] Zhang L, Dong D, Wang J. et al. A zinc finger protein SlSZP1 pro-tects SlSTOP1 from SlRAE1-mediated degradation to modulate aluminum resistance. New Phytol. 2022; 236:165-81

[30] Huang CF, Yamaji N, Mitani N. et al. A bacterial-type ABC transporter is involved in aluminum tolerance in rice. Plant Cell. 2009; 21:655-67

[31] Xu JM, Lou HQ, Jin JF. et al. A half-type ABC transporter FeS-TAR1 regulates Al resistance possibly via UDP-glucose-based hemicellulose metabolism and Al binding. Plant Soil. 2018; 432: 303-14

[32] Jin JF, Zhu HH, He QY. et al. The tomato transcription factor SlNAC063 is required for aluminum tolerance by regulating SlAAE3-1 expression. Front Plant Sci. 2022; 13:826954

[33] He QY, Jin JF, Li PF. et al. Involvement of SlSTOP1 regulated SlFDH expression in aluminum tolerance by reducing NAD+ to NADH in the tomato root apex. Plant J. 2023; 113:387-401

[34] Huang CF, Ma Y. Aluminum resistance in plants: a critical review focusing on STOP1. Plant Commun. 2024; 6:101200

[35] Maathuis FJM, Sanders D. Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana. Proc Natl Acad Sci USA. 1994; 91:9272-6

[36] Walker DJ, Leigh RA, Miller AJ. Potassium homeostasis in vacuo-late plant cells. Proc Natl Acad Sci USA. 1996; 93:10510-4

[37] Yang TY, Feng HM, Zhang S. et al. The potassium transporter OsHAK5 alters rice architecture via ATP-dependent transmem-brane auxin fluxes. Plant Commun. 2020; 1:100052

[38] Ye JY, Tian WH, Zhou M. et al. STOP1 activates NRT1.1-mediated nitrate uptake to create a favorable rhizospheric pH for plant adaptation to acidity. Plant Cell. 2021; 33:3658-74

[39] Balzergue C, Dartevelle T, Godon C. et al. Desnos T (2017) low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation. Nat Commun. 2017; 8:15300

[40] Xu JM, Wang ZQ, Wang JY. et al. Low phosphate represses histone deacetylase complex1 to regulate root system archi-tecture remodeling in Arabidopsis. New Phytol. 2020; 225: 1732-45

[41] Kollmeier M, Felle HH, Horst WJ. Genotypical differences in aluminum resistance of maize are expressed in the distal part of the transition zone. Is reduced basipetal auxin flow involved in inhibition of root elongation by aluminum. Plant Physiol. 2000; 122:945-56

[42] Yang ZB, Geng XY, He CM. et al. TAA1-regulated local auxin biosynthesis in the root-apex transition zone mediates the aluminum-induced inhibition of root growth in Arabidopsis. Plant Cell. 2014; 26:2889-904

[43] Spalding EP. Diverting the downhill flow of auxin to steer growth during tropisms. Am J Bot. 2013; 100:203-14

[44] Ahn SJ, Rengel Z, Matsumoto H. Aluminum-induced plasma membrane surface potential and H+-ATPase activity in near-isogenic wheat lines differing in tolerance to aluminum. New Phytol. 2004; 162:71-9

[45] Shen H, He LF, Sasaki T. et al. Citrate secretion coupled with the modulation of soybean root tip under aluminum stress.: up-regulation of transcription, translation, and threonine-oriented phosphorylation of plasma membrane H+-ATPase. Plant Physiol. 2005; 138:287-96

[46] Kim YS, Park W, Nian H. et al. Aluminum tolerance associated with enhancement of plasma membrane H+-ATPase in the root apex of soybean. Soil Sci Plant Nutr. 2010; 56:140-9

[47] Chen Q, Guo CL, Wang P. et al. Up-regulation and interaction of the plasma membrane H+-ATPase and the 14-3-3 protein are involved in the regulation of citrate exudation from the broad bean (Vicia faba L.) under Al stress. Plant Physiol Biochem. 2013; 70: 504-11

[48] Wang C, Bian C, Li J. et al. Melatonin promotes Al3+ compartmen-talization via H+ transport and ion gradients in Malus hupehensis. Plant Physiol. 2023; 193:821-39

[49] Zhu HH, Wang JY, Jiang D. et al. The miR157-SPL-CNR mod-ule acts upstream of bHLH101 to negatively regulate iron deficiency responses in tomato. J Integr Plant Biol. 2022; 64: 1059-75

[50] Barbez E, Dünser K, Gaidora A. et al. Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana. Proc Natl Acad Sci USA. 2017; 114:e4884-93