Calmodulin-like protein MdCML15 interacts with MdBT2 to modulate iron homeostasis in apple

Abstract

BTB and TAZ domain proteins (BTs) function as specialized adaptors facilitating substrate recognition of the CUL3-RING ubiquitin ligase (CRL3) complex that targets proteins for ubiquitination in reaction to diverse pressures. Nonetheless, knowledge of the molecular mechanisms by which the apple scaffold protein MdBT2 responds to external and internal signals is limited. Here we demonstrate that a putative Ca²⁺ sensor, calmodulin-like 15 (MdCML15), acts as an upstream regulator of MdBT2 to negatively modulate its functions in plasma membrane H⁺-ATPase regulation and iron deficiency tolerance. MdCML15 was identified to be substantially linked to MdBT2, and to result in the ubiquitination and degradation of the MdBT2 target protein MdbHLH104. Consequently, MdCML15 repressed the MdbHLH104 target, MdAHA8’s expression, reducing levels of a specific membrane H⁺-ATPase. Finally, the phenotype of transgenic apple plantlets and calli demonstrated that MdCML15 modulates membrane H⁺-ATPase-produced rhizosphere pH lowering alongside iron homeostasis through an MdCML15-MdBT2-MdbHLH104-MdAHA8 pathway. Our results provide new insights into the relationship between Ca²⁺ signaling and iron homeostasis.

Acknowledgements

We would like to sincerely thank our team leader, Dr Yu-Jin Hao. He will be remembered for his great achievement and for his support and assistance in this work. This work was supported by grants from National Key Research and Development Program (2022YFD1201701), the National Natural Science Foundation of China (32001336 and 32272683), the China Agriculture Research System of MOF and MARA (CARS-27), and the Natural Science Foundation of Shandong Province (ZR2022QC093).

Author contributions

C.X.Y and X.F.W. planned and designed the research. X.J.L., H.K., X.L., Q.Z., and Y.H.D. performed experiments and analyzed the data. Q.L., Y.X., and Y.X.Y. provided technical assistance. X.J.L., H.K., and X.F.W. wrote the article.

Data availability

The data underlying this article are available in the manuscript and in its online supplementary material.

Conflict of interest

The authors declare that no competing interests exist.

Supplementary data

Supplementary data are available at Horticulture Research online.

References

[1] Liang G. Iron uptake, signaling, and sensing in plants. Plant Commun. 2022; 3:100349

[2] Gao F, Dubos C. Transcriptional integration of plant responses to iron availability. J Exp Bot. 2021; 72:2056-70

[3] Santi S, Schmidt W. Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots. New Phytol. 2009; 183:1072-84

[4] Walker EL, Connolly EL. Time to pump iron: iron-deficiency-signaling mechanisms of higher plants. Curr Opin Plant Biol. 2008; 11:530-5

[5] Hindt MN, Guerinot ML. Getting a sense for signals: regulation of the plant iron deficiency response. BBA Mol Cell Res. 2012; 1823:1521-30

[6] Zhang X, Zhang D, Sun W. et al. The adaptive mechanism of plants to iron deficiency via iron uptake, transport, and homeostasis. Int J Mol Sci. 2019; 20:2424

[7] Brumbarova T, Bauer P, Ivanov R. Molecular mechanisms governing Arabidopsis iron uptake. Trends Plant Sci. 2015; 20:124-33

[8] Gao F, Robe K, Gaymard F. et al. The transcriptional control of iron homeostasis in plants: a tale of bHLH transcription factors? Front. Plant Sci. 2019; 10:6

[9] Yuan Y, Wu H, Wang N. et al. FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis. Cell Res. 2008; 18:385-97

[10] Wang N, Cui Y, Liu Y. et al. Requirement and functional redundancy of Ib subgroup bHLH proteins for iron deficiency responses and uptake in Arabidopsis thaliana. Mol Plant. 2013; 6:503-13

[11] Long TA, Tsukagoshi H, Busch W. et al. The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots. Plant Cell. 2010; 22:2219-36

[12] Zhang J, Liu B, Li M. et al. The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis. Plant Cell. 2015; 27:787-805

[13] Li X, Zhang H, Ai Q. et al. Two bHLH transcription factors, bHLH34 and bHLH104, regulate iron homeostasis in Arabidopsis thaliana. Plant Physiol. 2016; 170:2478-93

[14] Liang G, Zhang H, Li X. et al. bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana. J Exp Bot. 2017; 68:1743-55

[15] Kim SA, LaCroix IS, Gerber SA. et al. The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI. Proc Natl Acad Sci USA. 2019; 116:24933-42

[16] Gao F, Robe K, Bettembourg M. et al. The transcription factor bHLH121 interacts with bHLH105 (ILR3) and its closest homologs to regulate iron homeostasis in Arabidopsis. Plant Cell. 2020; 32:508-24

[17] Lei R, Li Y, Cai Y. et al. bHLH 121 functions as a direct link that facilitates the activation of FIT by bHLH IVc transcription factors for maintaining Fe homeostasis in Arabidopsis. Mol Plant. 2020; 13:634-49

[18] Zhao Q, Ren YR, Wang QJ. et al. Overexpression of MdbHLH104 gene enhances the tolerance to iron deficiency in apple. Plant Biotechnol J. 2016; 14:1633-45

[19] Fan Z, Wu Y, Zhao L. et al. MYB308-mediated transcriptional activation of plasma membrane H⁺-ATPase 6 promotes iron uptake in citrus. Hortic Res. 2022; 9:uhac088

[20] Selote D, Samira R, Matthiadis A. et al. Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors. Plant Physiol. 2015; 167:273-86

[21] Zhang HM, Li Y, Yao XN. et al. POSITIVE REGULATOR OF IRON HOMEOSTASIS 1 (OsPRI1) positively regulates iron homeostasis in rice. Plant Physiol. 2017; 175:543-54

[22] Gratz R, Brumbarova T, Ivanov R. et al. Phospho-mutant activity assays provide evidence for alternative phospho-regulation pathways of the transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR. New Phytol. 2020; 225:250-67

[23] Rodriguez-Celma J, Green RT, Connorton JM. et al. Arabidopsis BRUTUS-LIKE E 3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling. Proc Natl Acad Sci USA. 2019; 116:17584-91

[24] Zhao Q, Ren YR, Wang QJ. et al. Ubiquitination-related MdBT scaffold proteins target a bHLH transcription factor for iron homeostasis. Plant Physiol. 2016; 172:1973-88

[25] Zhou LJ, Zhang CL, Zhang RF. et al. The SUMO E3 ligase MdSIZ1 targets MdbHLH104 to regulate plasma membrane H⁺-ATPase activity and iron homeostasis. Plant Physiol. 2019; 179:88-106

[26] Li D, Sun Q, Zhang G. et al. MxMPK6-2-bHLH104 interaction is involved in reactive oxygen species signaling in response to iron deficiency in apple rootstock. J Exp Bot. 2021; 72:1919-32

[27] Zhai L, Sun Q, Gao M. et al. MxMPK4- 1 phosphorylates NADPH oxidase to trigger the MxMPK6-2-MxbHLH104 pathway mediated Fe deficiency responses in apple. Plant Cell Environ. 2022; 45:2810-26

[28] Xu L, Wei Y, Reboul J. et al. BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. Nature. 2003; 425:316-21

[29] Hua Z, Vierstra RD. The cullin-RING ubiquitin-protein ligases. Annu Rev Plant Bio. 2011; 62:299-334

[30] Robert HS, Quint A, Brand D. et al. BTB and TAZ domain scaffold proteins perform a crucial function in Arabidopsis development. Plant J. 2009; 58:109-21

[31] Du L, Poovaiah BW. A novel family of Ca²⁺/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. Plant Mol Biol. 2004; 54:549-69

[32] Figueroa P, Gusmaroli G, Serino G. et al. Arabidopsis has two redundant Cullin 3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo. Plant Cell. 2005; 17:1180-95

[33] Mandadi KK, Misra A, Ren S. et al. BT2, a BTB protein, mediates multiple responses to nutrients, stresses, and hormones in Arabidopsis. Plant Physiol. 2009; 150:1930-9

[34] Araus V, Vidal EA, Puelma T. et al. Members of BTB gene family of scaffold proteins suppress nitrate uptake and nitrogen use efficiency. Plant Physiol. 2016; 171:1523-32

[35] Irigoyen S, Ramasamy M, Misra A. et al. A BTB-TAZ protein is required for gene activation by cauliflower mosaic virus 35S multimerized enhancers. Plant Physiol. 2022; 188:397-410

[36] Ren S, Mandadi KK, Boedeker AL. et al. Regulation of telomerase in Arabidopsis by BT2, an apparent target of TELOMERASE ACTIVATOR1. Plant Cell. 2007; 19:23-31

[37] Uno Y, Milla MAR, Maher E. et al. Identification of proteins that interact with catalytically active calcium-dependent protein kinases from Arabidopsis. Mol Gen Genomics. 2009; 281:375-90

[38] An JP, Wang XF, Hao YJ. BTB/TAZ protein MdBT2 integrates multiple hormonal and environmental signals to regulate anthocyanin biosynthesis in apple. J Integr Plant Biol. 2020; 62:1643-6

[39] Kudla J, Becker D, Grill E. et al. Advances and current challenges in calcium signaling. New Phytol. 2018; 218:414-31

[40] Tong T, Li Q, Jiang W. et al. Molecular evolution of calcium signaling and transport in plant adaptation to abiotic stress. Int J Mol Sci. 2021; 22:12308

[41] Li T, Liu Z, Lv T. et al. Phosphorylation of MdCYTOKININ RESPONSE FACTOR4 suppresses ethylene biosynthesis during apple fruit ripening. Plant Physiol. 2023; 191:694-714

[42] Sun Q, Zhai L, Zhao D. et al. Kinase MxMPK4-1 and calmodulin-binding protein MxIQM3 enhance apple root acidification during Fe deficiency. Plant Physiol. 2023; 191:1968-84

[43] Zhang Q, Liu X, Liu X. et al. Genome-wide identification, characterization, and expression analysis of calmodulin-like proteins (CMLs) in apple. Hortic Plant J. 2017; 3:219-31

[44] Wang Z, Zhang Y, Liu Y. et al. Calcium-dependent protein kinases CPK21 and CPK23 phosphorylate and activate the iron-regulated transporter IRT1 to regulate iron deficiency in Arabidopsis. Sci China Life Sci. 2023; 66:2646-62

[45] Gratz R, Manishankar P, Ivanov R. et al. CIPK11-dependent phosphorylation modulates FIT activity to promote Arabidopsis iron acquisition in response to calcium signaling. Dev Cell. 2019; 48:726-740.e10

[46] Dobney S, Chiasson D, Lam P. et al. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J Biol Chem. 2009; 284:31647-57

[47] Zeng H, Xu L, Singh A. et al. Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses. Front. Plant Sci. 2015; 6:600

[48] Tsai YC, Koo Y, Delk NA. et al. Calmodulin-related CML24 interacts with ATG4b and affects autophagy progression in Arabidopsis. Plant J. 2013; 73:325-35

[49] Song X, Li J, Lyu M. et al. CALMODULIN-LIKE-38 and PEP1 RECEPTOR 2 integrate nitrate and brassinosteroid signals to regulate root growth. Plant Physiol. 2021; 187:1779-94

[50] Sun Q, Huang R, Zhu H. et al. A novel Medicago truncatula calmodulin-like protein (MtCML42) regulates cold tolerance and flowering time. Plant J. 2021; 108:1069-82

[51] Yu S, Wu J, Sun Y. et al. A calmodulin-like protein (CML10) interacts with cytosolic enzymes GSTU8 and FBA6 to regulate cold tolerance. Plant Physiol. 2022; 190:1321-33

[52] Wang XF, An JP, Liu X. et al. The nitrate-responsive protein MdBT2 regulates anthocyanin biosynthesis by interacting with the MdMYB1 transcription factor. Plant Physiol. 2018; 178:890-906

[53] Yang K, An JP, Li CY. et al. The apple C2H2-type zinc finger transcription factor MdZAT10 positively regulates JA-induced leaf senescence by interacting with MdBT2. Hortic Res. 2021; 8:159

[54] Ren YR, Zhao Q, Yang YY. et al. Interaction of BTB-TAZ protein MdBT2 and DELLA protein MdRGL3a regulates nitrate-mediated plant growth. Plant Physiol. 2021; 186:750-66

[55] Zhang QY, Gu KD, Cheng LL. et al. BTB-TAZ domain protein MdBT2 modulates malate accumulation and vacuolar acidification in response to nitrate. Plant Physiol. 2020; 183:750-64

[56] Zhang D, Yang K, Kan Z. et al. The regulatory module MdBT2-MdMYB88/MdMYB124-MdNRTs regulates nitrogen usage in apple. Plant Physiol. 2021; 185:1924-42

[57] Zhang QY, Gu KD, Wang JH. et al. BTB-BACK-TAZ domain protein MdBT2-mediated MdMYB73 ubiquitination negatively regulates malate accumulation and vacuolar acidification in apple. Hortic Res. 2020; 7:151

[58] Liu XJ, An XH, Liu X. et al. MdSnRK1.1 interacts with MdJAZ 18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation in apple. J Exp Bot. 2017; 68:2977-90

[59] Peretz Y, Mozes-Koch R, Akad F. et al. A universal expression/silencing vector in plants. Plant Physiol. 2007; 145:1251-63

[60] Xiao X, Ma F, Chen CL. et al. High efficient transformation of auxin reporter gene into trifoliate orange via Agrobacterium rhizogenes-mediated co-transformation. Plant Cell Tissue Organ Cult. 2014; 118:137-46

[61] Kang H, Zhang TT, Li YY. et al. The apple BTB protein MdBT2 positively regulates MdCOP1 abundance to repress anthocyanin biosynthesis. Plant Physiol. 2022; 190:305-18

[62] Sun WJ, Zhang JC, Ji XL. et al. Low nitrate alleviates iron deficiency by regulating iron homeostasis in apple. Plant Cell Environ. 2021; 44:1869-84

[63] Ren YR, Zhao Q, Yang YY. et al. The apple 14-3-3 protein MdGRF11 interacts with the BTB protein MdBT2 to regulate nitrate deficiency-induced anthocyanin accumulation. Hortic Res. 2021; 8:22