, Volume 248, Issue 4, pp 893–907 | Cite as

LeSPL-CNR negatively regulates Cd acquisition through repressing nitrate reductase-mediated nitric oxide production in tomato

  • Wei Wei Chen
  • Jian Feng Jin
  • He Qiang Lou
  • Li Liu
  • Leon V. Kochian
  • Jian Li YangEmail author
Original Article


Main conclusion

An SPL-type transcription factor, LeSPL-CNR, is negatively involved in NO production by modulating SlNR expression and nitrate reductase activity, which contributes to Cd tolerance.

Cadmium (Cd) is a highly toxic pollutant. Identifying factors affecting Cd accumulation in plants is a prerequisite for minimizing dietary uptake of Cd from crops grown with contaminated soil. Here, we report the involvement of a SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor LeSPL-CNR in Cd tolerance in tomato (Solanum lycopersicum). In comparison with the wild-type Ailsa Craig (AC) plants, the Colourless non-ripening (Cnr) epimutant displayed increased Cd accumulation and enhanced sensitivity to Cd, which was in well accordance with the repression of LeSPL-CNR expression. Cd stress-induced NO production was inhibited by nitrate reductase (NR) inhibitor, but not NO synthase-like enzyme inhibitor. Expression of LeSPL-CNR was negatively correlated with SlNR expression and the NR activity. We also demonstrated that LeSPL-CNR inhibited the SlNR promoter activity in vivo and bound to SlNR promoter sequence that does not contain a known SBP-binding motif. In addition, expression of an IRON-REGULATED TRANSPORTER1, SlIRT1, was more abundant in Cnr roots than AC roots under Cd stress. LeSPL-CNR may thus provide a molecular mechanism linking Cd stress response to regulation of NR-dependent NO production, which then contributes to Cd uptake via SlIRT1 expression in tomato.


Cadmium toxicity Iron uptake Solanum lycopersicum Transcription factor 



Ailsa Craig


Colourless non-ripening


4-Amino-5methylamino-2′,7′-difluorescein diacetate




Nitric oxide


Nitric oxide synthase


Nitrate reductase


Solanum lycopersicum nitrate reductase




Transcription factor



This work was supported financially by the Natural Science Foundation of China (31222049) and The Chang Jiang Scholars Program (JLY). We are grateful to Prof. Yiguo Hong (Hangzhou Normal University) for providing us the tomato seeds and his critical comments to the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary material

425_2018_2949_MOESM1_ESM.docx (2 mb)
Supplementary material 1 (DOCX 2020 kb)
425_2018_2949_MOESM2_ESM.xlsx (12 kb)
Supplementary material 2 (XLSX 12 kb)


  1. Abdel-Ghany SE, Pilon M (2008) MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis. J Biol Chem 283:15932–15945CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bernal M, Casero D, Singh V, Wilson GT, Grande A, Yang H, Dodani SC, Pellegrini M, Huijser P, Connolly EL, Merchant SS, Krämer U (2012) Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/FRO5 and the copper dependence of iron homeostasis in Arabidopsis. Plant Cell 224:738–761CrossRefGoogle Scholar
  3. Besson-Bard A, Gravot A, Richaud P, Auroy P, Duc C, Gaymard F, Taconnat L, Renou J-P, Pugin A, Wendehenne D (2009) Nitric oxide contributes to cadmium toxicity in Arabidopsis by promoting cadmium accumulation in roots and by up-regulating genes related to iron uptake. Plant Physiol 149:1302–1315CrossRefPubMedPubMedCentralGoogle Scholar
  4. Birkenbihl RP, Jach G, Saedler H, Huijser P (2005) Functional dissection of the plant-specific SBP-domain: overlap of the DNA binding and nuclear localization domains. J Mol Biol 352:585–596CrossRefPubMedGoogle Scholar
  5. Chen WW, Yang JL, Qin C, Jin CW, Mo JH, Ye T, Zheng SJ (2010a) Nitric oxide acts downstream of auxin to trigger root ferric chelate reductase activity in response to iron deficiency in Arabidopsis. Plant Physiol 154:810–819CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chen X, Zhang Z, Liu D, Zhang K, Li A, Mao L (2010b) SQUAMOSA promoter-binding protein-like transcription factors: star players for plant growth and development. J Integr Plant Biol 52:946–951CrossRefPubMedGoogle Scholar
  7. Chen W, Kong J, Qin C et al (2015a) Requirement of CHROMOMETHYLASE3 for somatic inheritance of the spontaneous tomato eipmutation Colourless non-ripening. Sci Rep 5:9192CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen W, Kong J, Lai T et al (2015b) Tuning LeSPL-CNR expression by SlymiR157 affects tomato fruit ripening. Sci Rep 5:7852. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chen J, Yang L, Yan X, Liu Y, Wang R, Fan T, Ren Y, Tang X, Xiao F, Liu Y (2016) Zinc-finger transcription factor ZAT6 positively regulates cadmium tolerance through the glutathione-dependent pathway in Arabidopsis. Plant Physiol 171:707–719CrossRefPubMedPubMedCentralGoogle Scholar
  10. Clemens S (2006) Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 88:1707–1719CrossRefPubMedGoogle Scholar
  11. DalCorso G, Farinati S, Maistri S, Furini A (2008) How plants cope with cadmium: stalking all on metabolism and gene expression. J Integr Plant Biol 50:1268–1280CrossRefPubMedGoogle Scholar
  12. Fan SK, Fang XZ, Guan MY, Ye YQ, Lin XY, Du ST, Jin CW (2014) Exogenous abscisic acid application decreases cadmium accumulation in Arabidopsis plants, which is associated with the inhibition of IRT1-mediated cadmium uptake. Front Plant Sci 5:721. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fan W, Lou HQ, Gong YL, Liu MY, Cao MJ, Liu Y, Yang JL, Zheng SJ (2015) Characterization of an inducible C2H2-type zinc finger transcription factor VuSOTP1 in rice bean (Vigna umbellata) reveals differential regulation between low pH and aluminum tolerance mechanisms. New Phytol 208:456–468CrossRefPubMedGoogle Scholar
  14. Farinati S, DalCorso G, Varotto S, Furini A (2010) The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants. New Phytol 185:964–978CrossRefPubMedGoogle Scholar
  15. Graziano M, Lamattina L (2007) Nitric oxide accumulation is required for molecular and physiological responses to iron deficiency in tomato roots. Plant J 52:949–960CrossRefPubMedGoogle Scholar
  16. Groppa MD, Rosales EP, Iannone MF, Benavides MP (2008) Nitric oxide, polyamines and Cd-induced phytotoxicity in wheat roots. Phytochemistry 69:2609–2615CrossRefPubMedGoogle Scholar
  17. Guo J-Y, Felippes FF, Liu C-J, Weigel D, Wang J-W (2011) Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 23:1512–1522CrossRefGoogle Scholar
  18. Guo H, Feng X, Hong C, Chen H, Zeng F, Zheng B, Jiang D (2017) Malate secretion from the root system is an important reason for higher resistance of Miscanthus sacchariflorus to cadmium. Physiol Plant 159:340–353CrossRefPubMedGoogle Scholar
  19. He XL, Fan SK, Zhu J, Guan MY, Liu XX, Zhang YS, Jin CW (2017) Iron supply prevents Cd uptake in Arabidopsis by inhibiting IRT1 expression and favoring competition between Fe and Cd uptake. Plant Soil 416:453–462CrossRefGoogle Scholar
  20. Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Laing WA (2005) Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1:13CrossRefPubMedPubMedCentralGoogle Scholar
  21. Jeandroz S, Wipf D, Stuehr DJ, Lamattina L, Melkonian M, Tian Z, Zhu Y, Carpenter EJ, Wong GK-S, Wendehenne D (2016) Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom. Sci Signal 9:2. CrossRefGoogle Scholar
  22. Jin CW, Du ST, Shamsi IH, Luo BF, Lin XY (2011) NO synthase-generated NO acts downstream of auxin in regulating Fe-deficiency-induced root branching that enhances Fe-deficiency tolerance in tomato plants. J Exp Bot 62:3875–3884CrossRefPubMedPubMedCentralGoogle Scholar
  23. Jung H, Gayomba SR, Rutzke MA, Craft E, Kochian LV, Vatamaniuk OK (2012) COPT6 is a plasma membrane transporter that functions in copper homeostasis in Arabidopsis and is a novel target of SQUAMOSA promoter-binding protein-like 7. J Biol Chem 287:33252–33267CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kan Q, Wu W, Yu W, Zhang J, Xu J, Rengel Z, Chen L, Cui X, Chen Q (2016) Nitrate reductase-mediated NO production enhances Cd accumulation in Panax notoginseng roots by affecting root cell wall properties. J Plant Physiol 193:64–70CrossRefPubMedGoogle Scholar
  25. Kim DY, Bovet L, Maeshima M, Martinoia E, Lee Y (2007) The ABC transporter AtPDR8 is a cadmium extrusion pump conferring heavy metal resistance. Plant J 50:207–218CrossRefPubMedGoogle Scholar
  26. Kim JJ, Lee JH, Kim W, Jung HS, Huijser P, Ahn JH (2012) The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambiet temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis. Plant Physiol 159:461–478CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kliebenstein DJ, Monde RA, Last RL (1998) Superoxide dismutase in Arabidopsis: an eclectic enzyme family with disparate regulation and protein localization. Plant Physiol 118:637–650CrossRefPubMedPubMedCentralGoogle Scholar
  28. Krzeslowska M (2011) The cell wall in plant cell response to trace metals: polysaccharide remodeling and its role in defense strategy. Acta Physiol Plant 33:35–51CrossRefGoogle Scholar
  29. Lamattina L, Garcia-Mata C, Graziano M, Pagnussat G (2003) Nitric oxide: the versatility of an extensive signal molecule. Annu Rev Plant Biol 54:109–136CrossRefPubMedGoogle Scholar
  30. Lei K-J, Lin Y-M, Ren J, Bai L, Miao Y-C, An G-Y, Song C-P (2015) Modulation of the phosphate-deficient responses by microRNA156 and its targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 in Arabidopsis. Plant Cell Physiol 57:192–203CrossRefPubMedGoogle Scholar
  31. Lux A, Martinka M, Vaculik M, White PJ (2011) Root responses to cadmium in the rhizosphere: a review. J Exp Bot 62:21–37CrossRefPubMedGoogle Scholar
  32. Manning K, Tor M, Poole M, Hong Y, Thompson AJ, King GJ, Giovannoni JJ, Seymour GB (2006) A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet 38:948–952CrossRefPubMedGoogle Scholar
  33. Mao QQ, Guan MY, Lu KX, Du ST, Fan SK, Ye YQ, Lin XY, Jin CW (2014) Inhibition of nitrate transporter 1.1-controlled nitrate uptake reduces cadmium uptake in Arabidopsis. Plant Physiol 166:934–944CrossRefPubMedPubMedCentralGoogle Scholar
  34. Mitsuda N, Ohme-Takagi M (2009) Functional analysis of transcription factors in Arabidopsis. Plant Cell Physiol 50:1232–1248CrossRefPubMedPubMedCentralGoogle Scholar
  35. Neill SJ, Desikan R, Hancock JT (2003) Nitric oxide signaling in plants. New Phytol 159:11–35CrossRefGoogle Scholar
  36. Preston JC, Hileman L (2013) Functional evolution in the plant SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) gene family. Front Plant Sci 4:80. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Romero-Puertas MC, Corpas FJ, Rodriguez-Serrano M, Gomez M, del Rio LA, Sandalio LM (2007) Differential expression and regulation of antioxidative enzymes by Cd in pea plants. J Plant Physiol 164:1346–1357CrossRefPubMedGoogle Scholar
  38. Shim D, Hwang JU, Lee J, Lee S, Choi Y, An G, Martinoia E, Lee Y (2009) Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice. Plant Cell 21:4031–4043CrossRefPubMedPubMedCentralGoogle Scholar
  39. Stone J, Liang X, Nekl E, Stiers J (2005) Arabidopsis AtSPL14, a plant-specific SBP-domain transcription factor, participates in plant development and sensitivity to fumonisin B1. Plant J 41:744–754CrossRefPubMedGoogle Scholar
  40. Sun C, Lu L, Liu L, Liu W, Yu Y, Liu X, Hu Y, Jin C, Lin X (2014) Nitrate reductase-mediated early nitric oxide burst alleviates oxidative damage induced by aluminum through enhancement of antioxidant defenses in roots of wheat (Triticum aestivum). New Phytol 201:1240–1250CrossRefPubMedGoogle Scholar
  41. Sunkar R, Kapoor A, Zhu JK (2006) Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by down-regulation of miR398 and important for oxidative stress tolerance. Plant Cell 18:2051–2065CrossRefPubMedPubMedCentralGoogle Scholar
  42. Tamas L, Dudikova J, Durcekova K, Haluskova L, Huttova J, Mistrık I, Olle M (2008) Alterations of the gene expression, lipid peroxidation, proline and thiol content along the barley root exposed to cadmium. J Plant Physiol 165:1193–1203CrossRefPubMedGoogle Scholar
  43. Tang QY, Zhang CX (2012) Data processing system (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research. Insect Sci 20:254–260CrossRefPubMedGoogle Scholar
  44. Thompson AJ, Tor M, Barry CS, Vrebalov J, OrWla C, Jarvis MC, Giovannoni JJ, Grierson D, Seymour GB (1999) Molecular and genetic characterization of a novel pleiotropic tomato-ripening mutant. Plant Physiol 120:383–390CrossRefPubMedPubMedCentralGoogle Scholar
  45. Ueno D, Yamaji N, Kono I, Huang CF, Ando T, Yano M, Ma JF (2010) Gene limiting cadmium accumulation in rice. Proc Natl Acad Sci USA 107:16500–16505CrossRefPubMedGoogle Scholar
  46. Valentovičová K, Halušková L, Huttová J, Mistrík I, Tamás L (2010) Effect of cadmium on diaphorase activity and nitric oxide production in barley root tips. J Plant Physiol 167:10–14CrossRefPubMedGoogle Scholar
  47. Verbruggen N, Hermans C, Schat H (2009) Mechanisms to cope with arsenic or cadmium excess in plants. Curr Opin Plant Biol 12:364–372CrossRefPubMedGoogle Scholar
  48. Weber M, Trampczynska A, Clemens S (2006) Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+-hypertolerant facultative metallophyte Arabidopsis halleri. Plant Cell Environ 29:950–963CrossRefPubMedGoogle Scholar
  49. Xiong J, Fu G, Tao L, Zhu C (2010) Roles of nitric oxide in alleviating heavy metal toxicity in plants. Arch Biochem Biophys 497:13–20CrossRefPubMedGoogle Scholar
  50. Xu J, Wang W, Yin H, Liu X, Sun H, Mi Q (2010) Exogenous nitric oxide improves antioxidative capacity and reduces auxin degradation in roots of Medicago truncatula seedlings under cadmium stress. Plant Soil 326:321–330CrossRefGoogle Scholar
  51. Yamasaki K, Kigawa T, Inoue M et al (2004) A novel zinc-binding motif revealed by solution structures of DNA-binding domains of Arabidopsis SBP family transcription factors. J Mol Biol 337:49–63CrossRefPubMedGoogle Scholar
  52. Yamasaki H, Abdel-Ghany SE, Cohu CM, Kobayashi Y, Shikanai T, Pilon M (2007) Regulation of copper homeostasis by micro-RNA in Arabidopsis. J Biol Chem 282:16369–16378CrossRefPubMedGoogle Scholar
  53. Yamasaki H, Hayashi M, Fukazawa M, Kobayashi Y, Shikanai T (2009) SQUAMOSA promoter binding protein-like 7 is a central regulator for copper homeostasis in Arabidopsis. Plant Cell 21:347–361CrossRefPubMedPubMedCentralGoogle Scholar
  54. Yuan H-M, Huang X (2016) Inhibition of root meristem growth by cadmium involves nitric oxide-mediated repression of auxin accumulation and signalling in Arabidopsis. Plant Cell Environ 39:120–135CrossRefPubMedGoogle Scholar
  55. Zhou Y, Xu XY, Chen LQ, Yang JL, Zheng SJ (2012) Nitric oxide exacerbates Al-induced inhibition of root elongation in rice bean by affecting cell wall and plasma membrane properties. Phytochemistry 76:46–51CrossRefPubMedGoogle Scholar
  56. Zhu XF, Zheng C, Hu YT, Jiang T, Liu Y, Dong NY, Yang JL, Zheng SJ (2011) Cadmium-induced oxalate secretion from root apex is associated with cadmium exclusion and resistance in Lycopersicon esculentum. Plant Cell Environ 34:1055–1064CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Research Centre for Plant RNA Signaling, College of Life and Environmental SciencesHangzhou Normal UniversityHangzhouChina
  2. 2.State Key Laboratory of Plant Physiology and Biochemistry, College of Life SciencesZhejiang UniversityHangzhouChina
  3. 3.State Key Laboratory of Subtropical Silviculture, School of Forestry and BiotechnologyZhejiang A&F UniversityLin’anChina
  4. 4.Global Institute for Food SecurityUniversity of SaskatchewanSaskatoonCanada

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