Skip to main content

DELLA proteins negatively regulate dark-induced senescence and chlorophyll degradation in Arabidopsis through interaction with the transcription factor WRKY6

Abstract

Key message

DELLA proteins’ negative regulation of dark-induced senescence and chlorophyll degradation in Arabidopsis is through interaction with WRKY6 and thus repression of its transcriptional activities on senescence-related genes.

Abstract

Senescence is an intricate and highly orchestrated process regulated by numerous endogenous and environmental signals. Gibberellins (GAs) and their signaling components DELLA proteins have been known to participate in the regulation of senescence. However, the mechanism of the GA-DELLA system involved in the senescence process remains largely unclear. Darkness is a known environmental factor that induces plant senescence. In this study, exogenous GA3 (an active form of GA) accelerated but paclobutrazol (a specific GA biosynthesis inhibitor) retarded dark-induced leaf yellowing in Arabidopsis. Moreover, the dark-triggered decrease in chlorophyll content, increase in cell membrane leakage, and upregulation of senescence-associated genes were notably impaired in both endogenous GA-decreased mutants ga3ox1/ga3ox2 and ga20ox1/ga20ox2 compared with those in wild-type Col-0. These effects of darkness were enhanced in the quintuple mutant of DELLA genes gai-t6/rga-t2/rgl1-1/rgl2-1/rgl3-1 and conversely attenuated in the gain-of-function mutant gai and transgenic plant 35S::TAP-RGAd17 compared with wild-type Ler. Subsequently, RGA interacted with the transcription factor WRKY6 in a yeast two-hybrid assay, as confirmed by bimolecular fluorescence complementation and pull-down analyses. In addition, mutation and overexpression of WRKY6 retarded and accelerated dark-induced senescence, respectively. Furthermore, transient expression assays in Arabidopsis protoplasts indicated that RGA and GAI weakened the transcriptional activities of WRKY6 on its downstream senescence-related genes, including SAG13 and SGR. Taken together, these results suggest that GAs positively and DELLAs negatively regulate dark-induced senescence and chlorophyll degradation in Arabidopsis. DELLAs function in this process, at least in part, by interacting with WRKY6.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

BiFC:

Bimolecular fluorescence complementation

della :

gai-t6/rga-t2/rgl1-1/rgl2-1/rgl3-1

fLUC:

Firefly luciferase

GA:

Gibberellin

GA3ox:

Gibberellin 3-oxidase

GA20ox:

Gibberellin 20-oxidase

GAI:

GA-insensitive

NYC1:

Non-yellow coloring 1

PAC:

Paclobutrazol

qRT-PCR:

Real-time quantitative reverse transcription polymerase chain reaction

REN LUC:

Renilla luciferase

RGA:

Repressor of ga1-3

SGR:

Stay green

SAG:

Senescence-associated gene

TF:

Transcription factor

References

  • Achard P, Liao L, Jiang C, Desnos T, Bartlett J, Fu X, Harberd NP (2007) DELLAs contribute to plant photomorphogenesis. Plant Physiol 143:1163–1172

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cheminant S, Wild M, Bouvier F, Pelletier S, Renou JP, Erhardt M, Hayes S, Terry MJ, Genschik P, Achard P (2011) DELLAs regulate chlorophyll and carotenoid biosynthesis to prevent photooxidative damage during seedling deetiolation in Arabidopsis. Plant Cell 23:1849–1860

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Chen YF, Li LQ, Xu Q, Kong YH, Wang H, Wu WH (2009) The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis. Plant Cell 21:3554–3566

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Chen M, Maodzeka A, Zhou L, Ali E, Wang Z, Jiang L (2014) Removal of DELLA repression promotes leaf senescence in Arabidopsis. Plant Sci 219–220:26–34

    Article  PubMed  CAS  Google Scholar 

  • Chen L, Xiang S, Chen Y, Li D, Yu D (2017) Arabidopsis WRKY45 interacts with the DELLA Protein RGL1 to positively regulate age-triggered leaf senescence. Mol Plant 10:1174–1189

    Article  PubMed  CAS  Google Scholar 

  • Davière JM, Achard P (2013) Gibberellin signaling in plants. Development 140:1147–1151

    Article  PubMed  CAS  Google Scholar 

  • Feng S, Martinez C, Gusmaroli G, Wang Y, Zhou J, Wang F, Chen L, Yu L, Iglesias-Pedraz JM, Kircher S, Schäfer E, Fu X, Fan LM, Deng XW (2008) Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature 451:475–479

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Goldthwaite JJ, Laetsch WM (1968) Control of senescence in rumex leaf discs by gibberellic acid. Plant Physiol 43:1855–1858

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Guo Y, Gan S (2005) Leaf senescence: signals, execution, and regulation. Curr Top Dev Biol 71:83–112

    Article  PubMed  CAS  Google Scholar 

  • Hörtensteiner S (2006) Chlorophyll degradation during senescence. Annu Rev Plant Biol 57:55–77

    Article  PubMed  CAS  Google Scholar 

  • Hörtensteiner S (2013) Update on the biochemistry of chlorophyll breakdown. Plant Mol Biol 82:505–517

    Article  PubMed  CAS  Google Scholar 

  • Kuai B, Chen J, Hörtensteiner S (2018) The biochemistry and molecular biology of chlorophyll breakdown. J Exp Bot 69:751–767

    Article  PubMed  Google Scholar 

  • Li QF, Wang C, Jiang L, Li S, Sun SS, He JX (2012) An interaction between BZR1 and DELLAs mediates direct signaling crosstalk between brassinosteroids and gibberellins in Arabidopsis. Sci Signal 5:ra72

    PubMed  Google Scholar 

  • Li Z, Peng J, Wen X, Guo H (2013) Ethylene-insensitive3 is a senescence-associated gene that accelerates age-dependent leaf senescence by directly repressing miR164 transcription in Arabidopsis. Plant Cell 25:3311–3328

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58:115–136

    Article  PubMed  CAS  Google Scholar 

  • Maymon I, Greenboim-Wainberg Y, Sagiv S, Kieber JJ, Moshelion M, Olszewski N, Weiss D (2009) Cytosolic activity of SPINDLY implies the existence of a DELLA-independent gibberellin-response pathway. Plant J 58:979–988

    Article  PubMed  CAS  Google Scholar 

  • Miao Y, Laun T, Zimmermann P, Zentgraf U (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol Biol 55:853–867

    Article  PubMed  CAS  Google Scholar 

  • Miao Y, Jiang J, Ren Y, Zhao Z (2013) The single-stranded DNA-binding protein WHIRLY1 represses WRKY53 expression and delays leaf senescence in a developmental stage-dependent manner in Arabidopsis. Plant Physiol 163:746–756

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mitchum MG, Yamaguchi S, Hanada A, Kuwahara A, Yoshioka Y, Kato T, Tabata S, Kamiya Y, Sun TP (2006) Distinct and overlapping roles of two gibberellin 3-oxidases in Arabidopsis development. Plant J 45:804–818

    Article  PubMed  CAS  Google Scholar 

  • Oh E, Zhu JY, Wang ZY (2012) Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat Cell Biol 14:802–809

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Plackett AR, Powers SJ, Fernandez-Garcia N, Urbanova T, Takebayashi Y, Seo M, Jikumaru Y, Benlloch R, Nilsson O, Ruiz-Rivero O, Phillips AL, Wilson ZA, Thomas SG, Hedden P (2012) Analysis of the developmental roles of the Arabidopsis gibberellin 20-oxidases demonstrates that GA20ox1, -2, and –3 are the dominant paralogs. Plant Cell 24:941–960

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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:e1005399

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ren Y, Li Y, Jiang Y, Wu B, Miao Y (2017) Phosphorylation of WHIRLY1 by CIPK14 shifts its localization and dual functions in Arabidopsis. Mol Plant 10:749–763

    Article  PubMed  CAS  Google Scholar 

  • Robatzek S, Somssich IE (2002) Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev 16:1139–1149

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 24:507–518

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sakuraba Y, Jeong J, Kang MY, Kim J, Paek NC, Choi G (2014) Phytochrome-interacting transcription factors PIF4 and PIF5 induce leaf senescence in Arabidopsis. Nat Commun 5:4636

    Article  PubMed  CAS  Google Scholar 

  • Shimoda Y, Ito H, Tanaka A (2016) Arabidopsis STAY-GREEN, Mendel’s green cotyledon gene, encodes magnesium-dechelatase. Plant Cell:2147–2160

  • Whyte P, Luckwill LC (1966) A sensitive bioassay for gibberellins based on retardation of leaf senescence in Rumex obtusifolius (L.). Nature 210:1360–1360

    Article  CAS  Google Scholar 

  • Wu FH, Shen SC, Lee LY, Lee SH, Chan MT, Lin CS (2009) Tape-Arabidopsis sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Xu H, Liu Q, Yao T, Fu X (2014) Shedding light on integrative GA signaling. Curr Opin Plant Biol 21:89–95

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi S (2008) Gibberellin metabolism and its regulation. Annu Rev Plant Biol 59:225–251

    Article  PubMed  CAS  Google Scholar 

  • Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

    Article  PubMed  CAS  Google Scholar 

  • Zhang H, Zhou C (2013) Signal transduction in leaf senescence. Plant Mol Biol 82:539–545

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y, Liu Z, Chen Y, He JX, Bi Y (2015a) PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) positively regulates dark-induced senescence and chlorophyll degradation in Arabidopsis. Plant Sci 237:57–68

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y, Liu Z, Wang J, Chen Y, Bi Y, He J (2015b) Brassinosteroid is required for sugar promotion of hypocotyl elongation in Arabidopsis in darkness. Planta 242:881–893

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y, Liu Z, Liu J, Lin S, Wang J, Lin W, Xu W (2017) GA-DELLA pathway is involved in regulation of nitrogen deficiency-induced anthocyanin accumulation. Plant Cell Rep 36:557–569

    Article  PubMed  CAS  Google Scholar 

  • Zhou X, Jiang Y, Yu D (2011) WRKY22 transcription factor mediates dark-induced leaf senescence in Arabidopsis. Mol Cells 31:303–313

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Dr. Peter Hedden for providing ga20ox1/ga20ox2 seeds, Dr. Nicholas P. Harberd for providing gai seeds, Dr. Xing-Wang Deng for providing 35S::TAP-RGAd17 seeds, Dr. Imre E. Somssich and Dr. Yi-Fang Chen for providing wrky6-1 and WRKY6OX seeds, and ABRC for providing della and ga3ox1/ga3ox2 seeds. This work was supported by National Natural Science Foundation of China (31701985 and 31700256), Natural Science Foundation of Fujian Province, China (2017J01611) and Fujian-Taiwan Joint Innovative Centre for Germplasm Resources and Cultivation of Crop (grant No. 2015-75. Fujian 2011 Program, China).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Yongqiang Zhang or Wenxiong Lin.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by Chun-Hai Dong.

Electronic supplementary material

Below is the link to the electronic supplementary material.

299_2018_2282_MOESM1_ESM.docx

Fig. S1 Effect of DELLAs on chlorophyll content and ion leakage in light-grown seedlings. Relative chlorophyll content and relative ion leakage were determined in 3-weeks-old light-grown seedlings of Ler, della, gai, and 35S::TAP-RGAd17. Fig. S2 Effect of darkness on expression of GA20OX1 and GA3OX1. After 3-weeks-old light-grown Col-0 seedlings were transferred to darkness for 0, 6, and 24 h, relative transcript levels of GA20OX1 and GA3OX1 were determined using qRT-PCR. (DOCX 22 KB)

Supplementary material 2 (DOCX 145 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, Y., Liu, Z., Wang, X. et al. DELLA proteins negatively regulate dark-induced senescence and chlorophyll degradation in Arabidopsis through interaction with the transcription factor WRKY6. Plant Cell Rep 37, 981–992 (2018). https://doi.org/10.1007/s00299-018-2282-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-018-2282-9

Keywords

  • Arabidopsis
  • Chlorophyll degradation
  • Dark
  • DELLA
  • Gibberellins
  • Senescence