The C-terminal cysteine-rich motif of NYE1/SGR1 is indispensable for its function in chlorophyll degradation in Arabidopsis

  • Zuokun Xie
  • Shengdong Wu
  • Junyi Chen
  • Xiaoyu Zhu
  • Xin Zhou
  • Stefan Hörtensteiner
  • Guodong RenEmail author
  • Benke KuaiEmail author

Key message

The C-terminal cysteine-rich motif of NYE1/SGR1 affects chlorophyll degradation likely by mediating its self-interaction and conformational change, and somehow altering its Mg-dechelating activity in response to the changing redox potential.


During green organ senescence in plants, the most prominent phenomenon is the degreening caused by net chlorophyll (Chl) loss. NON-YELLOWING1/STAY-GREEN1 (NYE1/SGR1) was recently reported to be able to dechelates magnesium (Mg) from Chl a to initiate its degradation, but little is known about the domain/motif basis of its functionality. In this study, we carried out a protein truncation assay and identified a conserved cysteine-rich motif (CRM, P-X3-C-X3-C-X-C2-F-P-X5-P) at its C terminus, which is essential for its function. Genetic analysis showed that all four cysteines in the CRM were irreplaceable, and enzymatic assays demonstrated that the mutation of each of the four cysteines affected its Mg-dechelating activity. The CRM plays a critical role in the conformational change and self-interaction of NYE1 via the formation of inter- and intra-molecular disulfide bonds. Our results may provide insight into how NYE1 responds to rapid redox changes during leaf senescence and in response to various environmental stresses.


Arabidopsis Chlorophyll degradation NYE1/SGR1 Cysteine-rich motif Redox regulation 



We are grateful to Jianxiang Liu for sharing pXY103 and pXY104 vectors, and Tongshui Zhou and Guojun Zhou for technical assistance on HPLC analysis. This work was supported by grants from the National Natural Science Foundation of China (31670287) to BK, the Science and Technology Commission of Shanghai Municipality (15JC1400800) to GR and the Swiss National Science Foundation (31003A_172977) to SH.

Author contributions

Conceived and designed the experiments: BK, GR, SH, ZX, JC. Performed the experiments: ZX, SW, JC, XZhu. Analyzed the data: ZX, JC. Contributed reagents/materials/analysis tools: XZho, SH. Wrote the paper: BK, GR, ZX, JC, SH, XZhu.


This work was supported by grants from the National Natural Science Foundation of China (31670287) to BK, the Science and Technology Commission of Shanghai Municipality (2015JC1400800) to GR, and the Swiss National Science Foundation (31003A_172977) to SH.

Supplementary material

11103_2019_902_MOESM1_ESM.docx (560 kb)
Supplementary material 1 (DOCX 559 kb)
11103_2019_902_MOESM2_ESM.docx (22 kb)
Supplementary material 2 (DOCX 22 kb)


  1. Armstead I, Donnison I, Aubry S, Harper J, Hortensteiner S, James C, Mani J, Moffet M, Ougham H, Roberts L, Thomas A, Weeden N, Thomas H, King I (2006) From crop to model to crop: identifying the genetic basis of the staygreen mutation in the Lolium/Festuca forage and amenity grasses. New Phytol 172:592–597CrossRefGoogle Scholar
  2. Armstead I, Donnison I, Aubry S, Harper J, Hortensteiner S, James C, Mani J, Moffet M, Ougham H, Roberts L, Thomas A, Weeden N, Thomas H, King I (2007) Cross-species identification of Mendel’s I locus. Science 315:73CrossRefGoogle Scholar
  3. Aroca A, Serna A, Gotor C, Romero LC (2015) S-sulfhydration: a cysteine posttranslational modification in plant systems. Plant Physiol 168:334–342CrossRefGoogle Scholar
  4. Aubry S, Mani J, Hortensteiner S (2008) Stay-green protein, defective in Mendel’s green cotyledon mutant, acts independent and upstream of pheophorbide a oxygenase in the chlorophyll catabolic pathway. Plant Mol Biol 67:243–256CrossRefGoogle Scholar
  5. Barry CS, McQuinn RP, Chung MY, Besuden A, Giovannoni JJ (2008) Amino acid substitutions in homologs of the STAY-GREEN protein are responsible for the green-flesh and chlorophyll retainer mutations of tomato and pepper. Plant Physiol 147:179–187CrossRefGoogle Scholar
  6. Borovsky Y, Paran I (2008) Chlorophyll breakdown during pepper fruit ripening in the chlorophyll retainer mutation is impaired at the homolog of the senescence-inducible stay-green gene. Theor Appl Genet 117:235–240CrossRefGoogle Scholar
  7. Chen J, Zhu X, Ren J, Qiu K, Li Z, Xie Z, Gao J, Zhou X, Kuai B (2017) Suppressor of overexpression of CO 1 negatively regulates dark-induced leaf degreening and senescence by directly repressing pheophytinase and other senescence-associated genes in Arabidopsis. Plant Physiol 173:1881–1891CrossRefGoogle Scholar
  8. Chen Y, Shimoda Y, Yokono M, Ito H, Tanaka A (2019) Mg-dechelatase is involved in the formation of photosystem II but not in chlorophyll degradation in Chlamydomonas reinhardtii. Plant J 97:1022–1031CrossRefGoogle Scholar
  9. Christ B, Schelbert S, Aubry S, Sussenbacher I, Muller T, Krautler B, Hortensteiner S (2012) MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis. Plant Physiol 158:628–641CrossRefGoogle Scholar
  10. Christ B, Sussenbacher I, Moser S, Bichsel N, Egert A, Muller T, Krautler B, Hortensteiner S (2013) Cytochrome P450 CYP89A9 is involved in the formation of major chlorophyll catabolites during leaf senescence in Arabidopsis. Plant Cell 25:1868–1880CrossRefGoogle Scholar
  11. Christ B, Egert A, Sussenbacher I, Krautler B, Bartels D, Peters S, Hortensteiner S (2014) Water deficit induces chlorophyll degradation via the ‘PAO/phyllobilin’ pathway in leaves of homoio- (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants. Plant Cell Environ 37:2521–2531CrossRefGoogle Scholar
  12. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefGoogle Scholar
  13. Delmas F, Sankaranarayanan S, Deb S, Widdup E, Bournonville C, Bollier N, Northey JG, McCourt P, Samuel MA (2013) ABI3 controls embryo degreening through Mendel’s I locus. Proc Natl Acad Sci USA 110:E3888–E3894CrossRefGoogle Scholar
  14. Fang C, Li C, Li W, Wang Z, Zhou Z, Shen Y, Wu M, Wu Y, Li G, Kong LA, Liu C, Jackson SA, Tian Z (2014) Concerted evolution of D1 and D2 to regulate chlorophyll degradation in soybean. Plant J 77:700–712CrossRefGoogle Scholar
  15. Gao S, Gao J, Zhu X, Song Y, Li Z, Ren G, Zhou X, Kuai B (2016) ABF2, ABF3, and ABF4 promote ABA-mediated chlorophyll degradation and leaf senescence by transcriptional activation of chlorophyll catabolic genes and senescence-associated genes in Arabidopsis. Mol Plant 9:1272–1285CrossRefGoogle Scholar
  16. Ge X, Dietrich C, Matsuno M, Li G, Berg H, Xia Y (2005) An Arabidopsis aspartic protease functions as an anti-cell-death component in reproduction and embryogenesis. EMBO Rep 6:282–288CrossRefGoogle Scholar
  17. Giles NM, Watts AB, Giles GI, Fry FH, Littlechild JA, Jacob C (2003) Metal and redox modulation of cysteine protein function. Chem Biol 10:677–693CrossRefGoogle Scholar
  18. Gray J, Close PS, Briggs SP, Johal GS (1997) A novel suppressor of cell death in plants encoded by the Lls1 gene of maize. Cell 89:25–31CrossRefGoogle Scholar
  19. Gray J, Wardzala E, Yang M, Reinbothe S, Haller S, Pauli F (2004) A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers. Plant Mol Biol 54:39–54CrossRefGoogle Scholar
  20. Hauenstein M, Christ B, Das A, Aubry S, Hortensteiner S (2016) A role for TIC55 as a hydroxylase of phyllobilins, the products of chlorophyll breakdown during plant senescence. Plant Cell 28:2510–2527CrossRefGoogle Scholar
  21. Hortensteiner S (2009) Stay-green regulates chlorophyll and chlorophyll-binding protein degradation during senescence. Trends Plant Sci 14:155–162CrossRefGoogle Scholar
  22. Jiang H, Li M, Liang N, Yan H, Wei Y, Xu X, Liu J, Xu Z, Chen F, Wu G (2007) Molecular cloning and function analysis of the stay green gene in rice. Plant J 52:197–209CrossRefGoogle Scholar
  23. Khanna-Chopra R (2012) Leaf senescence and abiotic stresses share reactive oxygen species-mediated chloroplast degradation. Protoplasma 249:469–481CrossRefGoogle Scholar
  24. Krautler B (2014) Phyllobilins—the abundant bilin-type tetrapyrrolic catabolites of the green plant pigment chlorophyll. Chem Soc Rev 43:6227–6238CrossRefGoogle Scholar
  25. Kuai B, Chen J, Hortensteiner S (2018) The biochemistry and molecular biology of chlorophyll breakdown. J Exp Bot 69:751–767CrossRefGoogle Scholar
  26. Li S, Gao J, Yao L, Ren G, Zhu X, Gao S, Qiu K, Zhou X, Kuai B (2016) The role of ANAC072 in the regulation of chlorophyll degradation during age- and dark-induced leaf senescence. Plant Cell Rep 35:1729–1741CrossRefGoogle Scholar
  27. Li Z, Wu S, Chen J, Wang X, Gao J, Ren G, Kuai B (2017) NYEs/SGRs-mediated chlorophyll degradation is critical for detoxification during seed maturation in Arabidopsis. Plant J 92:650–661CrossRefGoogle Scholar
  28. Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58:115–136CrossRefGoogle Scholar
  29. Luo Z, Zhang J, Li J, Yang C, Wang T, Ouyang B, Li H, Giovannoni J, Ye Z (2013) A STAY-GREEN protein SlSGR1 regulates lycopene and beta-carotene accumulation by interacting directly with SlPSY1 during ripening processes in tomato. New Phytol 198:442–452CrossRefGoogle Scholar
  30. Matsuda K, Shimoda Y, Tanaka A, Ito H (2016) Chlorophyll a is a favorable substrate for Chlamydomonas Mg-dechelatase encoded by STAY-GREEN. Plant Physiol Biochem 109:365–373CrossRefGoogle Scholar
  31. Mecey C, Hauck P, Trapp M, Pumplin N, Plovanich A, Yao J, He SY (2011) A critical role of STAYGREEN/Mendel’s I locus in controlling disease symptom development during Pseudomonas syringae pv tomato infection of Arabidopsis. Plant Physiol 157:1965–1974CrossRefGoogle Scholar
  32. Meguro M, Ito H, Takabayashi A, Tanaka R, Tanaka A (2011) Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis. Plant Cell 23:3442–3453CrossRefGoogle Scholar
  33. Mou Z, Fan W, Dong X (2003) Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell 113:935–944CrossRefGoogle Scholar
  34. Mur LA, Aubry S, Mondhe M, Kingston-Smith A, Gallagher J, Timms-Taravella E, James C, Papp I, Hortensteiner S, Thomas H, Ougham H (2010) Accumulation of chlorophyll catabolites photosensitizes the hypersensitive response elicited by Pseudomonas syringae in Arabidopsis. New Phytol 188:161–174CrossRefGoogle Scholar
  35. Pan J, Tan J, Wang Y, Zheng X, Owens K, Li D, Li Y, Weng Y (2018) STAYGREEN (CsSGR) is a candidate for the anthracnose (Colletotrichum orbiculare) resistance locus cla in Gy14 cucumber. Theor Appl Genet 131:1577–1587CrossRefGoogle Scholar
  36. Park SY, Yu JW, Park JS, Li J, Yoo SC, Lee NY, Lee SK, Jeong SW, Seo HS, Koh HJ, Jeon JS, Park YI, Paek NC (2007) The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19:1649–1664CrossRefGoogle Scholar
  37. Pruzinska A, Tanner G, Anders I, Roca M, Hortensteiner S (2003) Chlorophyll breakdown: pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene. Proc Natl Acad Sci USA 100:15259–15264CrossRefGoogle Scholar
  38. Pruzinska A, Anders I, Aubry S, Schenk N, Tapernoux-Luthi E, Muller T, Krautler B, Hortensteiner S (2007) In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown. Plant Cell 19:369–387CrossRefGoogle Scholar
  39. Qian L, Voss-Fels K, Cui Y, Jan HU, Samans B, Obermeier C, Qian W, Snowdon RJ (2016) Deletion of a stay-green gene associates with adaptive selection in Brassica napus. Mol Plant 9:1559–1569CrossRefGoogle Scholar
  40. Qiu K, Li Z, Yang Z, Chen J, Wu S, Zhu X, Gao S, Gao J, Ren G, Kuai B, Zhou X (2015) EIN3 and ORE1 accelerate degreening during ethylene-mediated leaf senescence by directly activating chlorophyll catabolic genes in Arabidopsis. PLoS Genet 11:e1005399CrossRefGoogle Scholar
  41. Ren G, An K, Liao Y, Zhou X, Cao Y, Zhao H, Ge X, Kuai B (2007) Identification of a novel chloroplast protein AtNYE1 regulating chlorophyll degradation during leaf senescence in Arabidopsis. Plant Physiol 144:1429–1441CrossRefGoogle Scholar
  42. Ren G, Zhou Q, Wu S, Zhang Y, Zhang L, Huang J, Sun Z, Kuai B (2010) Reverse genetic identification of CRN1 and its distinctive role in chlorophyll degradation in Arabidopsis. J Integr Plant Biol 52:496–504Google Scholar
  43. Rissler HM, Collakova E, Dellapenna D, Whelan J, Pogson BJ (2002) Chlorophyll biosynthesis. Expression of a second chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis. Plant Physiol 128:770–779CrossRefGoogle Scholar
  44. Sakuraba Y, Schelbert S, Park SY, Han SH, Lee BD, Andres CB, Kessler F, Hortensteiner S, Paek NC (2012) STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis. Plant Cell 24:507–518CrossRefGoogle Scholar
  45. Sakuraba Y, Jeong J, Kang MY, Kim J, Paek NC, Choi G (2014a) Phytochrome-interacting transcription factors PIF4 and PIF5 induce leaf senescence in Arabidopsis. Nat Commun 5:4636CrossRefGoogle Scholar
  46. Sakuraba Y, Kim D, Kim YS, Hortensteiner S, Paek NC (2014b) Arabidopsis STAYGREEN-LIKE (SGRL) promotes abiotic stress-induced leaf yellowing during vegetative growth. FEBS Lett 588:3830–3837CrossRefGoogle Scholar
  47. Sakuraba Y, Park SY, Kim YS, Wang SH, Yoo SC, Hortensteiner S, Paek NC (2014c) Arabidopsis STAY-GREEN2 is a negative regulator of chlorophyll degradation during leaf senescence. Mol Plant 7:1288–1302CrossRefGoogle Scholar
  48. Sato Y, Morita R, Nishimura M, Yamaguchi H, Kusaba M (2007) Mendel’s green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway. Proc Natl Acad Sci USA 104:14169–14174CrossRefGoogle Scholar
  49. Sato Y, Morita R, Katsuma S, Nishimura M, Tanaka A, Kusaba M (2009) Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice. Plant J 57:120–131CrossRefGoogle Scholar
  50. Schelbert S, Aubry S, Burla B, Agne B, Kessler F, Krupinska K, Hortensteiner S (2009) Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 21:767–785CrossRefGoogle Scholar
  51. Shimoda Y, Ito H, Tanaka A (2016) Arabidopsis STAY-GREEN, Mendel’s green cotyledon gene, encodes magnesium-dechelatase. Plant Cell 28:2147–2160CrossRefGoogle Scholar
  52. Song Y, Yang C, Gao S, Zhang W, Li L, Kuai B (2014) Age-triggered and dark-induced leaf senescence require the bHLH transcription factors PIF3, 4, and 5. Mol Plant 7:1776–1787CrossRefGoogle Scholar
  53. Wang Y, Yun BW, Kwon E, Hong JK, Yoon J, Loake GJ (2006) S-nitrosylation: an emerging redox-based post-translational modification in plants. J Exp Bot 57:1777–1784CrossRefGoogle Scholar
  54. Wang X, Gao J, Gao S, Song Y, Yang Z, Kuai B (2019) The H3K27me3 demethylase REF6 promotes leaf senescence through directly activating major senescence regulatory and functional genes in Arabidopsis. PLoS Genet 15:e1008068CrossRefGoogle Scholar
  55. Wu S, Li Z, Yang L, Xie Z, Chen J, Zhang W, Liu T, Gao S, Gao J, Zhu Y, Xin J, Ren G, Kuai B (2016) NON-YELLOWING2 (NYE2), a close paralog of NYE1, plays a positive role in chlorophyll degradation in Arabidopsis. Mol Plant 9:624–627CrossRefGoogle Scholar
  56. Zhou C, Han L, Pislariu C, Nakashima J, Fu C, Jiang Q, Quan L, Blancaflor EB, Tang Y, Bouton JH, Udvardi M, Xia G, Wang ZY (2011) From model to crop: functional analysis of a STAY-GREEN gene in the model legume Medicago truncatula and effective use of the gene for alfalfa improvement. Plant Physiol 157:1483–1496CrossRefGoogle Scholar
  57. Zhou J, Wang J, Cheng Y, Chi YJ, Fan B, Yu JQ, Chen Z (2013) NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. PLoS Genet 10:e1004477Google Scholar
  58. Zhu X, Chen J, Xie Z, Gao J, Ren G, Gao S, Zhou X, Kuai B (2015) Jasmonic acid promotes degreening via MYC2/3/4- and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes. Plant J 84:597–610CrossRefGoogle Scholar
  59. Zhu X, Chen J, Qiu K, Kuai B (2017) Phytohormone and light regulation of chlorophyll degradation. Front Plant Sci 8:1911CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Zuokun Xie
    • 1
    • 2
  • Shengdong Wu
    • 1
    • 2
  • Junyi Chen
    • 1
    • 2
  • Xiaoyu Zhu
    • 1
    • 2
  • Xin Zhou
    • 1
    • 2
  • Stefan Hörtensteiner
    • 3
  • Guodong Ren
    • 1
    • 2
    Email author
  • Benke Kuai
    • 1
    • 2
    Email author
  1. 1.State Key Laboratory of Genetic Engineering, School of Life SciencesFudan UniversityShanghaiChina
  2. 2.Ministry of Education Key Laboratory for Biodiversity Science and Ecological EngineeringInstitute of Biodiversity Science, Fudan UniversityShanghaiChina
  3. 3.Institute of Plant and Microbial Biology, University of ZurichZurichSwitzerland

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