NIGT1 family proteins exhibit dual mode DNA recognition to regulate nutrient response-associated genes in Arabidopsis


Autoři: Yoshiaki Ueda aff001;  Shohei Nosaki aff003;  Yasuhito Sakuraba aff001;  Takuya Miyakawa aff003;  Takatoshi Kiba aff004;  Masaru Tanokura aff003;  Shuichi Yanagisawa aff001
Působiště autorů: Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan aff001;  Crop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki, Japan aff002;  Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan aff003;  Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan aff004;  Center for Sustainable Resource Science, RIKEN, Tsurumi, Yokohama, Japan aff005
Vyšlo v časopise: NIGT1 family proteins exhibit dual mode DNA recognition to regulate nutrient response-associated genes in Arabidopsis. PLoS Genet 16(11): e32767. doi:10.1371/journal.pgen.1009197
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009197

Souhrn

Fine-tuning of nutrient uptake and response is indispensable for maintenance of nutrient homeostasis in plants, but the details of underlying mechanisms remain to be elucidated. NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) family proteins are plant-specific transcriptional repressors that function as an important hub in the nutrient signaling network associated with the acquisition and use of nitrogen and phosphorus. Here, by yeast two-hybrid assays, bimolecular fluorescence complementation assays, and biochemical analysis with recombinant proteins, we show that Arabidopsis NIGT1 family proteins form a dimer via the interaction mediated by a coiled-coil domain (CCD) in their N-terminal regions. Electrophoretic mobility shift assays defined that the NIGT1 dimer binds to two different motifs, 5'-GAATATTC-3' and 5'-GATTC-N38-GAATC-3', in target gene promoters. Unlike the dimer of wild-type NIGT1 family proteins, a mutant variant that could not dimerize due to amino acid substitutions within the CCD had lower specificity and affinity to DNA, thereby losing the ability to precisely regulate the expression of target genes. Thus, expressing the wild-type and mutant NIGT1 proteins in the nigt1 quadruple mutant differently modified NIGT1-regulated gene expression and responses towards nitrate and phosphate. These results suggest that the CCD-mediated dimerization confers dual mode DNA recognition to NIGT1 family proteins, which is necessary to make proper controls of their target genes and nutrient responses. Intriguingly, two 5'-GATTC-3' sequences are present in face-to-face orientation within the 5'-GATTC-N38-GAATC-3' sequence or its complementary one, while two 5'-ATTC-3' sequences are present in back-to-back orientation within the 5'-GAATATTC-3' or its complementary one. This finding suggests a unique mode of DNA binding by NIGT1 family proteins and may provide a hint as to why target sequences for some transcription factors cannot be clearly determined.

Klíčová slova:

Arabidopsis thaliana – Dimerization – DNA-binding proteins – Gene expression – Nitrates – Protein interactions – Seedlings – Sequence motif analysis


Zdroje

1. Bloom AJ, Chapin FS, Mooney HA. Resource limitation in plants-An economic analogy. Annu Rev Ecol Evol Syst. 1985;16:363–392.

2. Richardson AE, Barea JM, McNeill AM, Prigent-Combaret C. Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant Soil. 2009;321:305–339.

3. Kirk GJD, Kronzucker HJ. The potential for nitrification and nitrate uptake in the rhizosphere of wetland plants: A modelling study. Ann Bot. 2005;96:639–646. doi: 10.1093/aob/mci216 16024557

4. Raghothama KG. Phosphate transport and signaling. Curr Opin Plant Biol. 2000;3:182–187. 10837272

5. Bustos R, Castrillo G, Linhares F, Puga MI, Rubio V, Pérez-Pérez J, et al. A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis. PLoS Genet. 2010;6:e1001102. doi: 10.1371/journal.pgen.1001102 20838596

6. Nilsson L, Müller R, Nielsen TH. Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant, Cell Environ. 2007;30:1499–1512. doi: 10.1111/j.1365-3040.2007.01734.x 17927693

7. Makino A. Photosynthesis, grain yield, and nitrogen utilization in rice and wheat. Plant Physiol. 2011;155:125–129. doi: 10.1104/pp.110.165076 20959423

8. Vinod KK, Heuer S. Approaches towards nitrogen- and phosphorus-efficient rice. AoB Plants. 2012;2012: pls028. doi: 10.1093/aobpla/pls028 23115710

9. Xu G, Fan X, Miller AJ. Plant nitrogen assimilation and use efficiency. Annu Rev Plant Biol. 2012;63:153–182. doi: 10.1146/annurev-arplant-042811-105532 22224450

10. Ueda Y, Ohtsuki N, Kadota K, Tezuka A, Nagano AJ, Kadowaki T, et al. Gene regulatory network and its constituent transcription factors that control nitrogen deficiency responses in rice. New Phytol. 2020;227:1434–1452. doi: 10.1111/nph.16627 32343414

11. Konishi M, Yanagisawa S. Arabidopsis NIN-like transcription factors have a central role in nitrate signalling. Nat Commun. 2013;4:1617. doi: 10.1038/ncomms2621 23511481

12. Zhou J, Jiao F, Wu Z, Li Y, Wang X, He X, et al. OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants. Plant Physiol. 2008;146:1673–1686. doi: 10.1104/pp.107.111443 18263782

13. Wang J, Sun J, Miao J, Guo J, Shi Z, He M, et al. A phosphate starvation response regulator Ta-PHR1 is involved in phosphate signalling and increases grain yield in wheat. Ann Bot. 2013;111:1139–1153. doi: 10.1093/aob/mct080 23589634

14. Rubio V, Linhares F, Solano R, Martín AC, Iglesias J, Leyva A, et al. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev. 2001;15:2122–2133.

15. Qi W, Manfield IW, Muench SP, Baker A. AtSPX1 affects the AtPHR1–DNA-binding equilibrium by binding monomeric AtPHR1 in solution. Biochem J. 2017;474:3675–3687. doi: 10.1042/BCJ20170522 28887383

16. Osorio MB, Ng S, Berkowitz O, De Clercq I, Mao C, Shou H, et al. SPX4 acts on PHR1-dependent and -independent regulation of shoot phosphorus status in Arabidopsis. Plant Physiol. 2019;181:332–352. doi: 10.1104/pp.18.00594 31262954

17. Puga MI, Mateos I, Charukesi R, Wang Z, Franco-Zorrilla JM, De Lorenzo L, et al. SPX1 is a phosphate-dependent inhibitor of PHOSPHATE STARVATION RESPONSE 1 in Arabidopsis. Proc Natl Acad Sci U S A. 2014;111:14947–14952. doi: 10.1073/pnas.1404654111 25271326

18. Wild R, Gerasimaite R, Jung JY, Truffault V, Pavlovic I, Schmidt A, et al. Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science. 2016;352:986–990. doi: 10.1126/science.aad9858 27080106

19. Lv Q, Zhong Y, Wang Y, Wang Z, Zhang L, Shi J, et al. SPX4 negatively regulates phosphate signaling and homeostasis through its interaction with PHR2 in rice. Plant Cell. 2014;26:1586–1597. doi: 10.1105/tpc.114.123208 24692424

20. Nishida H, Tanaka S, Handa Y, Ito M, Sakamoto Y, Matsunaga S, et al. A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus. Nat Commun. 2018;9:499.

21. Wang M, Hasegawa T, Hayashi M, Ohmori Y, Yano K, Teramoto S, et al. OsNLP4 is required for nitrate assimilation gene expressions and nitrate-dependent growth in rice. bioRxiv. 2020. doi: 10.1101/2020.03.16.993733

22. Marchive C, Roudier F, Castaings L, Bréhaut V, Blondet E, Colot V, et al. Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants. Nat Commun. 2013;4:1713. doi: 10.1038/ncomms2650 23591880

23. Konishi M, Yanagisawa S. Emergence of a new step towards understanding the molecular mechanisms underlying nitrate-regulated gene expression. J Exp Bot. 2014;65:5589–5600. doi: 10.1093/jxb/eru267 25005135

24. Maeda Y, Konishi M, Kiba T, Sakuraba Y, Sawaki N, Kurai T, et al. A NIGT1-centred transcriptional cascade regulates nitrate signalling and incorporates phosphorus starvation signals in Arabidopsis. Nat Commun. 2018;9:1376. doi: 10.1038/s41467-018-03832-6 29636481

25. Liu KH, Niu Y, Konishi M, Wu Y, Du H, Sun Chung H, et al. Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks. Nature. 2017;545:311–316. doi: 10.1038/nature22077 28489820

26. Kiba T, Inaba J, Kudo T, Ueda N, Konishi M, Mitsuda N, et al. Repression of nitrogen starvation responses by members of the arabidopsis GARP-type transcription factor NIGT1/HRS1 subfamily. Plant Cell. 2018;30:925–945. doi: 10.1105/tpc.17.00810 29622567

27. Sawaki N, Tsujimoto R, Shigyo M, Konishi M, Toki S, Fujiwara T, et al. A nitrate-inducible GARP family gene encodes an auto-repressible transcriptional repressor in rice. Plant Cell Physiol. 2013;54:506–517. doi: 10.1093/pcp/pct007 23324170

28. Medici A, Marshall-Colon A, Ronzier E, Szponarski W, Wang R, Gojon A, et al. AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip. Nat Commun. 2015;6:6274. doi: 10.1038/ncomms7274 25723764

29. Liu H, Yang H, Wu C, Feng J, Liu X, Qin H, et al. Overexpressing HRS1 confers hypersensitivity to low phosphate-elicited inhibition of primary root growth in Arabidopsis thaliana. J Integr Plant Biol. 2009;51:382–392. doi: 10.1111/j.1744-7909.2009.00819.x 19341407

30. Ueda Y, Kiba T, Yanagisawa S. Nitrate-inducible NIGT1 proteins modulate phosphate uptake and starvation signalling via transcriptional regulation of SPX genes. Plant J. 2020;102:448–466. doi: 10.1111/tpj.14637 31811679

31. Ueda Y, Yanagisawa S. Perception, transduction, and integration of nitrogen and phosphorus nutritional signals in the transcriptional regulatory network in plants. J Exp Bot. 2019;70:3709–3717. doi: 10.1093/jxb/erz148 30949701

32. Yanagisawa S. Characterization of a nitrate-inducible transcriptional repressor NIGT1 provides new insights into DNA recognition by the GARP family proteins. Plant Signal Behav. 2013;8:e24447. doi: 10.4161/psb.24447 23603966

33. Hosoda K, Imamura A, Katoh E, Hatta T, Tachiki M, Yamada H, et al. Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators. Plant Cell. 2002;14:2015–2029. doi: 10.1105/tpc.002733 12215502

34. Ruan W, Guo M, Xu L, Wang X, Zhao H, Wang J, et al. An SPX-RLI1 module regulates leaf inclination in response to phosphate availability in rice. Plant Cell. 2018;30:853–870. doi: 10.1105/tpc.17.00738 29610209

35. Waters MT, Wang P, Korkaric M, Capper RG, Saunders NJ, Langdale JA. GLK transcription factors coordinate expression of the photosynthetic apparatus in Arabidopsis. Plant Cell. 2009;21:1109–1128. doi: 10.1105/tpc.108.065250 19376934

36. Safi A, Medici A, Szponarski W, Ruffel S, Lacombe B, Krouk G. The world according to GARP transcription factors. Curr Opin Plant Biol. 2017;39: 159–167. doi: 10.1016/j.pbi.2017.07.006 28802165

37. O’Shea EK, Rutkowski R, Kim PS. Evidence that the leucine zipper is a coiled coil. Science. 1989;243:538–542. doi: 10.1126/science.2911757 2911757

38. Landschulz WH, Johnson PF, McKnight SL. The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science. 1988;240:1759–1764. doi: 10.1126/science.3289117 3289117

39. Mason JM, Arndt KM. Coiled coil domains: Stability, specificity, and biological implications. ChemBioChem. 2004;5:170–176. doi: 10.1002/cbic.200300781 14760737

40. Maekawa S, Ishida T, Yanagisawa S. Reduced expression of APUM24, encoding a novel rRNA processing factor, induces sugar-dependent nucleolar stress and altered sugar responses in Arabidopsis thaliana. Plant Cell. 2018;30:209–227. doi: 10.1105/tpc.17.00778 29242314

41. Das M, Kobayashi M, Yamada Y, Sreeramulu S, Ramakrishnan C, Wakatsuki S, et al. Design of disulfide-linked thioredoxin dimers and multimers through analysis of crystal contacts. J Mol Biol. 2007;372:1278–1292. doi: 10.1016/j.jmb.2007.07.033 17727880

42. Shin H, Shin HS, Dewbre GR, Harrison MJ. Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. Plant J. 2004;39:629–642. doi: 10.1111/j.1365-313X.2004.02161.x 15272879

43. Alonso-Blanco C, Andrade J, Becker C, Bemm F, Bergelson J, Borgwardt KMM, et al. 1,135 genomes reveal the global pattern of polymorphism in Arabidopsis thaliana. Cell. 2016;166:481–491. doi: 10.1016/j.cell.2016.05.063 27293186

44. Mansueto L, Fuentes RR, Borja FN, Detras J, Abrio-Santos JM, Chebotarov D, et al. Rice SNP-seek database update: New SNPs, indels, and queries. Nucleic Acids Res. 2017;45:D1075–D1081. doi: 10.1093/nar/gkw1135 27899667

45. Konishi M, Yanagisawa S. The role of protein-protein interactions mediated by the PB1 domain of NLP transcription factors in nitrate-inducible gene expression. BMC Plant Biol. 2019;19:90. doi: 10.1186/s12870-019-1692-3 30819094

46. Boer DR, Freire-Rios A, Van Den Berg WAM, Saaki T, Manfield IW, Kepinski S, et al. Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors. Cell. 2014;156:577–589. doi: 10.1016/j.cell.2013.12.027 24485461

47. Chen WF, Wei X-B, Rety S, Huang LY, Liu NN, Dou SX, et al. Structural analysis reveals a “molecular calipers” mechanism for a LATERAL ORGAN BOUNDARIES DOMAIN transcription factor protein from wheat. J Biol Chem. 2019;294:142–156. doi: 10.1074/jbc.RA118.003956 30425099

48. Falvo J V., Lin CH, Tsytsykova A V., Hwang PK, Thanos D, Goldfeld AE, et al. A dimer-specific function of the transcription factor NFATp. Proc Natl Acad Sci U S A. 2008;105: 19637–19642. doi: 10.1073/pnas.0810648105 19060202

49. Amoutzias GD, Robertson DL, Van de Peer Y, Oliver SG. Choose your partners: dimerization in eukaryotic transcription factors. Trends Biochem Sci. 2008;33:220–229. doi: 10.1016/j.tibs.2008.02.002 18406148

50. Umesono K, Murakami KK, Thompson CC, Evans RM. Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell. 1991;65:1255–1266. doi: 10.1016/0092-8674(91)90020-y 1648450

51. Zechel C, Shen XQ, Chambon P, Gronemeyer H. Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements. EMBO J. 1994;13:1414–1424. 8137825

52. Al-Zyoud WA, Hynson RMG, Ganuelas LA, Coster ACF, Duff AP, Baker MAB, et al. Binding of transcription factor GabR to DNA requires recognition of DNA shape at a location distinct from its cognate binding site. Nucleic Acids Res. 2016;44:1411–1420. doi: 10.1093/nar/gkv1466 26681693

53. Oakley MG, Kim PS. A buried polar interaction can direct the relative orientation of helices in a coiled coil. Biochemistry. 1998;37:12603–12610. doi: 10.1021/bi981269m 9730833

54. Kapinos LE, Burkhard P, Herrmann H, Aebi U, Strelkov S V. Simultaneous formation of right- and left-handed anti-parallel coiled-coil interfaces by a Coil2 fragment of human lamin A. J Mol Biol. 2011;408:135–146. doi: 10.1016/j.jmb.2011.02.037 21354179

55. Yan Y, Shen L, Chen Y, Bao S, Thong Z, Yu H. A MYB-domain protein EFM mediates flowering responses to environmental cues in Arabidopsis. Dev Cell. 2014;30:437–448. doi: 10.1016/j.devcel.2014.07.004 25132385

56. Moreau F, Thévenon E, Blanvillain R, Lopez-Vidriero I, Franco-Zorrilla JM, Dumas R, et al. The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis. Dev. 2016;143:1108–1119. doi: 10.1242/dev.127365 26903506

57. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003;301:653–657. doi: 10.1126/science.1086391 12893945

58. Murashige T, Skoog F. A revised medium for rapid growth and bio assays with Tobacco tissue cultures. Physiol Plant. 1962;15:474–497.

59. Ward JT, Lahner B, Yakubova E, Salt DE, Raghothama KG. The effect of iron on the primary root elongation of Arabidopsis during phosphate deficiency. Plant Physiol. 2008;147:1181–1191. doi: 10.1104/pp.108.118562 18467463

60. Mora-Macías J, Ojeda-Rivera JO, Gutiérrez-Alanís D, Yong-Villalobos L, Oropeza-Aburto A, Raya-González J, et al. Malate-dependent Fe accumulation is a critical checkpoint in the root developmental response to low phosphate. Proc Natl Acad Sci U S A. 2017;114:E3563–E3572. doi: 10.1073/pnas.1701952114 28400510

61. Balzergue C, Dartevelle T, Godon C, Laugier E, Meisrimler C, Teulon JM, et al. Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation. Nat Commun. 2017;8:15300. doi: 10.1038/ncomms15300 28504266

62. Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, Taheri-Talesh N, et al. Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc. 2006;1:3111–3120. doi: 10.1038/nprot.2006.405 17406574

63. Tanaka Y, Kimura T, Hikino K, Goto S, Nishimura M, Mano S, et al. Gateway vectors for plant genetic engineering: overview of plant vectors, application for bimolecular fluorescence complementation (BiFC) and multigene construction. In: Barrera-Saldaña HA, editor. Genetic Engineering—Basics, New Applications and Responsibilities. Rijeka: InTechOpen; 2012.

64. Gietz RD, Schiestl RH. Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007;2:1–4. doi: 10.1038/nprot.2007.17 17401330

65. Sparkes IA, Runions J, Kearns A, Hawes C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc. 2006;1:2019–25. doi: 10.1038/nprot.2006.286 17487191

66. Ueda Y, Siddique S, Frei M. A novel gene, OZONE-RESPONSIVE APOPLASTIC PROTEIN1, enhances cell death in ozone stress in rice. Plant Physiol. 2015;169:873–889. doi: 10.1104/pp.15.00956 26220952

67. Shah KH, Almaghrabi B, Bohlmann H. Comparison of expression vectors for transient expression of recombinant proteins in plants. Plant Mol Biol Report. 2013;31:1529–1538. doi: 10.1007/s11105-013-0614-z 24415845

68. Liu L, Zhang Y, Tang S, Zhao Q, Zhang Z, Zhang H, et al. An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana. Plant J. 2010;61:893–903. doi: 10.1111/j.1365-313X.2009.04109.x 20015064

69. Saleh A, Alvarez-Venegas R, Avramova Z. An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc. 2008;3:1018–1025. doi: 10.1038/nprot.2008.66 18536649

70. Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M. Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell. 2000;12:393–404. doi: 10.1105/tpc.12.3.393 10715325

71. Yoo S-D, Cho Y-H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007;2:1565–1572. doi: 10.1038/nprot.2007.199 17585298

72. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16:735–743.

73. Kanno S, Cuyas L, Javot H, Bligny R, Gout E, Dartevelle T, et al. Performance and limitations of phosphate quantification: guidelines for plant biologists. Plant Cell Physiol. 2016;57:690–706. doi: 10.1093/pcp/pcv208 26865660

74. Lupas A, Van Dyke M, Stock J. Predicting coiled coils from protein sequences. Science. 1991;252:1162–1164. doi: 10.1126/science.252.5009.1162 2031185

75. de Castro E, Sigrist CJA, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res. 2006;34:W362–W365. doi: 10.1093/nar/gkl124 16845026

76. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–425 doi: 10.1093/oxfordjournals.molbev.a040454 3447015

77. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–1874. doi: 10.1093/molbev/msw054 27004904


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