Advertisement

Reverse genetics analysis of the 55-kDa B regulatory subunit of 2A serine/threonine protein phosphatase (PP2A) related to self-incompatibility in Chinese cabbage

  • Gi-Ho Lee
  • Na-Ri Shin
  • Young-Doo ParkEmail author
Research Report
  • 19 Downloads

Abstract

The type 2A serine/threonine protein phosphatases (PP2As) are key components in regulating signal transduction and controlling cell metabolism. In particular, PP2A has a role in pollen-pistil interaction during pollination in Brassica. In this study, reverse genetics screening was used to obtain a T-DNA-inserted Brassica rapa mutant line with the self-compatible phenotype. The type 2A PP2A 55-kDa B regulatory subunit gene (PR55/B) in this mutant was knocked out by T-DNA insertion, and its expression level was decreased, which led to self-compatible phenotypes, such as seed setting by flower self-pollination. For functional analysis of this self-compatible mutant line, the full-length PR55/B gene related to the self-incompatibility mechanism was isolated and the down-regulation vector (pPPi) was constructed for introduction into Chinese cabbage. Chinese cabbage lines transformed with pPPi showed significantly decreased PR55/B mRNA accumulation, which yielded self-compatible phenotypes. These transgenic lines were found to set pods and seeds by the self-pollination in both buds and flowers. Down-regulation of PP2A, which is related to self-incompatibility signal transduction, led to higher levels of the phosphorylated S-locus receptor kinase (SRK) substrate, which phosphorylated SRK blocks activation of armadillo-repeat-containing 1 (ARC1). Thus, the self-incompatibility response by ARC1 was not activated, and this led to the self-compatible response. Finally, PP2A is proposed to be a significant factor in self-incompatibility of Chinese cabbage.

Keywords

Brassica rapa Flower-pollination Insertional mutant line Reverse genetics Self-incompatibility 

Notes

Acknowledgements

This work was carried out with the support of the “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01365201)”, Rural Development Administration, Republic of Korea.

Author contributions

G-HL performed the majority of the experiment and data analysis. N-RS contributed to fertility experiment and data analysis. Y-DP designed the experiment and analyzed data. G-HL and Y-DP wrote manuscript. All authors contributed to and corrected the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13580_2019_194_MOESM1_ESM.docx (159 kb)
Supplementary material 1 (DOCX 159 kb)

References

  1. Alonso JM, Ecker JR (2006) Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis. Nat Rev Genet 7:524–536.  https://doi.org/10.1038/nrg1893 CrossRefPubMedGoogle Scholar
  2. Bower MS, Matias DD, Fernandes-Carvalho E, Mazzurco M, Gu T, Rothstein SJ et al (1996) Two members of the thioredoxin-h family interacts with the kinase domain of a Brassica S locus receptor kinase. Plant Cell 8:1647–1650.  https://doi.org/10.2307/3870256 CrossRefGoogle Scholar
  3. Boyes DC, Nasrallah JB (1993) Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea. Mol Gen Genet 236:369–373.  https://doi.org/10.1007/bf00277135 CrossRefPubMedGoogle Scholar
  4. Braun DM, Stone JM, Walker JC (1997) Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases: implications for transmembrane signalling in plants. Plant J 12:83–95.  https://doi.org/10.1046/j.1365-313x.1997.12010083.x CrossRefPubMedGoogle Scholar
  5. Cabrillac D, Cock JM, Dumas C, Gaude T (2001) The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins. Nature 410:220–223.  https://doi.org/10.1038/35065626 CrossRefPubMedGoogle Scholar
  6. Cohen P, Holmes CFB, Tsukitani Y (1990a) Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci 15:98–102.  https://doi.org/10.1016/0968-0004(90)90192-e CrossRefPubMedGoogle Scholar
  7. Cohen PT, Brewis ND, Hughes V, Mann DJ (1990b) Protein serine/threonine phosphatases; an expanding family. FEBS Lett 268:355–359.  https://doi.org/10.1016/0014-5793(90)81285-V CrossRefPubMedGoogle Scholar
  8. Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A (2006) Opposing activities protect against age-onset proteotoxicity. Science 313:1604–1610.  https://doi.org/10.1126/science.1124646 CrossRefPubMedGoogle Scholar
  9. Dixit R, Nasrallah JB (2001) Recognizing self in the self-incompatibility response. Plant Physiol 125:105–108.  https://doi.org/10.1104/pp.125.1.105 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fobis-Loisy I, Miege C, Gaude T (2004) Molecular evolution of the s locus controlling mating in the brassicaceae. Plant Biol 6:109–118.  https://doi.org/10.1055/s-2004-817804 CrossRefPubMedGoogle Scholar
  11. Franklin-Tong VE, Franklin FC (2000) Self-incompatibility in Brassica: the elusive pollen S gene is identified! Plant Cell 12:305–308.  https://doi.org/10.1105/tpc.12.3.305 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M et al (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92–100.  https://doi.org/10.1126/science.1068275 CrossRefPubMedGoogle Scholar
  13. Goring DR, Rothstein SJ (1992) The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase. Plant Cell 4:1273–1281.  https://doi.org/10.1105/tpc.4.10.1273 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Goring DR, Walker JC (2004) Plant sciences. Self-rejection—a new kinase connection. Science 303:1474–1475.  https://doi.org/10.1126/science.1095764 CrossRefPubMedGoogle Scholar
  15. Gu T, Mazzurco M, Sulaman W, Matias DD, Goring DR (1998) Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase. Proc Natl Acad Sci USA 95:382–387.  https://doi.org/10.1073/pnas.95.1.382 CrossRefPubMedGoogle Scholar
  16. Haffani YZ, Silva NF, Goring DR (2004) Receptor kinase signalling in plants. Can J Bot 82:1–15.  https://doi.org/10.1139/b03-126 CrossRefGoogle Scholar
  17. Haynes JG, Hartung AJ, Hendershot JD 3rd, Passingham RS, Rundle SJ (1999) Molecular characterization of the B’ regulatory subunit gene family of Arabidopsis protein phosphatase 2A. Eur J Biochem 260:127–136.  https://doi.org/10.1104/pp.020004 CrossRefPubMedGoogle Scholar
  18. Janssens V, Goris J (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353:417–439.  https://doi.org/10.1042/bj3530417 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Jyotishwaran G, Kotresha D, Selvaraj T, Srideshikan SM, Rajvanshi PK, Jayabaskaran C (2007) A modified freeze-thaw method for efficient transformation of Agrobacterium tumefaciens. Curr Sci 93:770–772Google Scholar
  20. Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195.  https://doi.org/10.1016/s1360-1385(02)02251-3 CrossRefPubMedGoogle Scholar
  21. Kim HS, Kim SH, Park YD (2003) Development of rescue cloning vector with phosphinothricin resistant gene for effective T-DNA tagging. Hortic Sci Technol 44:407–411Google Scholar
  22. Kusaba M, Dwyer K, Hendershot J, Vrebalov J, Nasrallah JB, Nasrallah ME (2001) Self-incompatibility in the genus Arabidopsis: characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana. Plant Cell 13:627–643.  https://doi.org/10.2307/3871411 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lee MK, Kim HS, Kim JS, Kim SH, Park YD (2004) Agrobacterium-mediated transformation system for large-scale producion of transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants for insertional mutagenesis. J Plant Biol 47:300–306.  https://doi.org/10.1007/bf03030544 CrossRefGoogle Scholar
  24. Li W, Gao B, Lee SM, Bennett K, Fang D (2007) RLE-1, an E3 ubiquitin ligase, regulates C. elegans aging by catalyzing DAF-16 polyubiquitination. Dev Cell 12:235–246.  https://doi.org/10.1016/j.devcel.2006.12.002 CrossRefPubMedGoogle Scholar
  25. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and 2^[-delta deltaC(T)]method. Methods 25:402–408.  https://doi.org/10.1006/meth.2001.1262 CrossRefGoogle Scholar
  26. Nakanishi T, Hinata K (1972) An effective time for CO2 gas treatment in overcoming self-incompatibility in Brassica. Plant Cell Physiol 14:873–879.  https://doi.org/10.1093/oxfordjournals.pcp.a074929 CrossRefGoogle Scholar
  27. Nakanishi T, Esashi Y, Hinata K (1969) Control of self-incompatibility by CO2 gas in Brassica. Plant Cell Physiol 10:925–927.  https://doi.org/10.1093/oxfordjournals.pcp.a074478 CrossRefGoogle Scholar
  28. Nasrallah JB (2000) Cell-cell signaling in the self-incompatibility response. Curr Opin Plant Biol 3:368–373.  https://doi.org/10.1016/s1369-5266(00)00098-4 CrossRefPubMedGoogle Scholar
  29. Nasrallah JB (2002) Recognition and rejection of self in plant reproduction. Science 296:305–308.  https://doi.org/10.1126/science.296.5566.305 CrossRefPubMedGoogle Scholar
  30. Park KH, Park HT, Chung CG (2010) Recent trends of the seed industry and a strategy to foster domestic seed companies. Korea Rural Economic Institute report p129Google Scholar
  31. Rundle SJ, Hartung AJ, Corum JW 3rd, O’Neill M (1995) Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A. Plant Mol Biol 28:257–266.  https://doi.org/10.1007/bf00020245 CrossRefPubMedGoogle Scholar
  32. Schierup MH, Mable BK, Awadalla P, Charlesworth D (2001) Identification and characterization of a polymorphic receptor kinase gene linked to the self-incompatibility locus of Arabidopsis lyrata. Genetics 158:387–399PubMedPubMedCentralGoogle Scholar
  33. Scutt CP, Fordham-Skelton AP, Croy RRD (1993) Okadaic acid causes breakdown of self-incompatibility in Brassica oleracea: evidence for the involvement of protein phosphatases in the incompatible response. Sex Plant Reprod 6:282–285.  https://doi.org/10.1007/bf00231906 CrossRefGoogle Scholar
  34. Shivanna KR, Heslop-Harrison Y, Heslop-Harrison J (1978) The pollen stigma interaction: bud pollination in Cruciferae. Acta Bot Neerl 27:107–119.  https://doi.org/10.1111/j.1438-8677.1978.tb00265.x CrossRefGoogle Scholar
  35. Smith RD, Walker JC (1996) Plant protein phosphatases. Annu Rev Plant Physiol Plant Mol Biol 47:101–125.  https://doi.org/10.1146/annurev.arplant.47.1.101 CrossRefPubMedGoogle Scholar
  36. Stein JC, Nasrallah JB (1993) A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L., encodes a functional serine/threonine kinase. Plant Physiol 101:1103–1106.  https://doi.org/10.1104/pp.101.3.1103 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB (1991) Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc Natl Acad Sci USA 88:8816–8820.  https://doi.org/10.1073/pnas.88.19.8816 CrossRefPubMedGoogle Scholar
  38. Stone SL, Arnoldo M, Goring DR (1999) A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants. Science 286:1729–1731.  https://doi.org/10.1126/science.286.5445.1729 CrossRefPubMedGoogle Scholar
  39. Stone SL, Anderson EM, Mullen RT, Goring DR (2003) ARC1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen. Plant Cell 15:885–898.  https://doi.org/10.1105/tpc.009845 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Syntichaki P, Troulinaki K, Tavernarakis N (2007) eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans. Nature 445:922–926.  https://doi.org/10.1038/nature05603 CrossRefPubMedGoogle Scholar
  41. Takayama S, Isogai A (2005) Self-incompatibility in plants. Annu Rev Plant Biol 56:467–489.  https://doi.org/10.1146/annurev.arplant.56.032604.144249 CrossRefPubMedGoogle Scholar
  42. Takayama S, Shiba H, Iwano M, Shimosato H, Che F-S, Kai N et al (2000) The pollen determinant of self-incompatibility in Brassica campestris. Proc Natl Acad Sci USA 97:1920–1925.  https://doi.org/10.1073/pnas.040556397 CrossRefPubMedGoogle Scholar
  43. Takayama S, Shimosato H, Shiba H, Funato M, Che F-S, Watanabe M et al (2001) Direct ligand–receptor complex interaction controls Brassica self-incompatibility. Nature 413:534–538.  https://doi.org/10.1038/35097104 CrossRefPubMedGoogle Scholar
  44. Tang C, Toomajian C, Sherman-Broyles S, Plagnol V, Guo YL, Hu TT et al (2007) The evolution of selfing in Arabidopsis thaliana. Science 317:1070–1072.  https://doi.org/10.1126/science.1143153 CrossRefPubMedGoogle Scholar
  45. Tao G, Yang R (1986) Use of CO2 and salt solution to overcome self-incompatibility of Chinese cabbage (B. campestris ssp. pekinensis). Eucarpia Crucif Newsl 11:75–76Google Scholar
  46. Terol J, Bargues M, Carrasco P, Pérez-Alonso M, Paricio N (2002) Molecular characterization and evolution of the protein phosphatase 2A B’ regulatory subunit family in plants. Plant Physiol 129:808–822.  https://doi.org/10.1104/pp.020004 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Thakore CU, Livengood AJ, Hendershot JD, Corum JW, LaTorre KA, Rundle SJ (1999) Characterization of the promoter region and expression pattern of three Arabidopsis protein phosphatase type 2A subunit genes. Plant Sci 147:165–176.  https://doi.org/10.1016/s0168-9452(99)00111-9 CrossRefGoogle Scholar
  48. Tichtinsky G, Vanoosthuyse V, Cock JM, Gaude T (2003) Making inroads into plant receptor kinase signalling pathways. Trends Plant Sci 8:231–237.  https://doi.org/10.1016/S1360-1385(03)00062-1 CrossRefPubMedGoogle Scholar
  49. Tlngdong F, Ping S, Xiaoniu Y, Guangsheng Y (1992) Overcoming self-incompatibility of Brassica napus by salt (NaCl) spray. Plant Breed 109:255–258.  https://doi.org/10.1111/j.1439-0523.1992.tb00181.x CrossRefGoogle Scholar
  50. Vanoosthuyse V, Tichtinsky G, Dumas C, Gaude T, Cock JM (2003) Interaction of calmodulin, a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase. Plant Physiol 133:919–929.  https://doi.org/10.1104/pp.103.023846 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Wolff S, Dillin A (2006) The trifecta of aging in Caenorhabditis elegans. Exp Gerontol 41:894–903.  https://doi.org/10.1016/j.exger.2006.06.054 CrossRefPubMedGoogle Scholar
  52. Yin YF, Baggett JR, Rowe KE (1981) The effects of bud self-pollination and open flower self-pollination on the field characteristics of broccoli (Brassica oleracea var. Italica). Euphytica 30:841–845.  https://doi.org/10.1007/bf00038813 CrossRefGoogle Scholar
  53. Yu JG, Lee GH, Kim JS, Shim EJ, Park YD (2010a) An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA. Mol Cells 29:267–275.  https://doi.org/10.1007/s10059-010-0013-3 CrossRefPubMedGoogle Scholar
  54. Yu JG, Lee GH, Lim KB, Hwang YJ, Woo ET, Kim JS et al (2010b) Analysis of mutant Chinese cabbage plants using gene tagging system. Hortic Sci Technol 28:442–448Google Scholar
  55. Yu JG, Lee GH, Park YD (2012) Comparison of RNA interference-mediated gene silencing and T-DNA integration techniques for gene function analysis in Chinese cabbage. Hortic Sci Technol 30:734–742.  https://doi.org/10.7235/hort.2012.12093 CrossRefGoogle Scholar

Copyright information

© Korean Society for Horticultural Science 2019

Authors and Affiliations

  1. 1.Department of Horticultural BiotechnologyKyung Hee UniversityYongin-siRepublic of Korea

Personalised recommendations