Plant Molecular Biology

, Volume 79, Issue 6, pp 609–622 | Cite as

RAP2.6L overexpression delays waterlogging induced premature senescence by increasing stomatal closure more than antioxidant enzyme activity

  • Peiqing Liu
  • Feng Sun
  • Rong Gao
  • Hansong Dong


Waterlogging usually results from overuse or poor management of irrigation water and is a serious constraint due to its damaging effects. RAP2.6L (At5g13330) overexpression enhances plant resistance to jasmonic acid, salicylic acid, abscisic acid (ABA) and ethylene in Arabidopsis thaliana. However, it is not known whether RAP2.6L overexpression in vivo improves plant tolerance to waterlogging stress. In this study, the RAP2.6L transcript was induced by waterlogging or an ABA treatment, which was reduced after pretreatment with an ABA biosynthesis inhibitor tungstate. Water loss and membrane leakage were reduced in RAP2.6L overexpression plants under waterlogging stress. Time course analyses of ABA content and production of hydrogen peroxide (H2O2) showed that increased ABA precedes the increase of H2O2. It is also followed by a marked increase in the antioxidant enzyme activities. Increased ABA promoted stomatal closure and made leaves exhibit a delayed waterlogging induced premature senescence. Furthermore, RAP2.6L overexpression caused significant increases in the transcripts of antioxidant enzyme genes APX1 (ascorbate peroxidase 1) and FSD1 (Fe-superoxide dismutase 1), the ABA biosynthesis gene ABA1 (ABA deficient 1) and signaling gene ABH1 (ABA-hypersensitive 1) and the waterlogging responsive gene ADH1 (alcohol dehydrogenase 1), while the transcript of ABI1 (ABA insensitive 1) was decreased. ABA inhibits seed germination and seedling growth and phenotype analysis showed that the integration of abi1-1 mutation into the RAP2.6L overexpression lines reduces ABA sensitivity. These suggest that RAP2.6L overexpression delays waterlogging induced premature senescence and might function through ABI1-mediated ABA signaling pathway.


RAP2.6L ABA Waterlogging Stomatal closure Antioxidant enzyme activity ABA signaling 



This study was supported by Key Basic Scientific Study and Development Plan (973 plan; grant no. 2012CB114003) and Natural Science Foundation (grant no. 31171830) of China, and Jiangsu Provincial Priority Academic Program Development of Higher Education Institutions.

Supplementary material

11103_2012_9936_MOESM1_ESM.jpg (472 kb)
Fig. S1 Molecular characterization of mutants and RAP2.6L overexpression plants. (A) Scheme of the transformation unit containing 35S promoter and RAP2.6L -GFP sequence. (B) Transcripts of RAP2.6L in mutants and RAP2.6L overexpression plants. Total RNA was prepared from 3-week-old WT, GFP, mutants and RAP2.6L overexpression plants. Transcripts were determined by qRT-PCR. The y axis is presented on a logarithmic scale for better comparison of fold changes. (C) Expression patterns of RAP2.6L in WT, GFP and RAP2.6L overexpression plants. Total RNA was prepared from the leaves of 3-week-old WT, GFP and RAP2.6L overexpression plants. Transcripts were determined by qRT-PCR. (D) Protein gel blot shows the expression level of RAP2.6L with GFP fusion proteins using GFP antibody in WT and RAP2.6L overexpression plants. Values are means ±SE (n=6). Means denoted by the same letter did not significantly differ at P <0.05 according to Student’s t test. (JPEG 472 kb)
11103_2012_9936_MOESM2_ESM.jpg (574 kb)
Fig. S2 Localization of the RAP2.6L protein in plants. Localization of RAP2.6L in GFP and RAP2.6L overexpression plants. Roots of RAP2.6L overexpression seedlings were used for examination using a laser-scanning confocal microscope (Bio-Rad MRC 1024). Scale bars: 20µM. OE: RAP2.6L RAP2.6L overexpression plants; 121GFP: empty vector RAP2.6L overexpression plants. (JPEG 573 kb)
11103_2012_9936_MOESM3_ESM.jpg (1 mb)
Fig. S3 RAP2.6L overexpression accelerates flowering in Arabidopsis. Seeds were sown on MS medium and incubated for 2 days at 4 oC in darkness to break dormancy before transferring to the growth chamber. Flowering phenotype was recorded after 18 days in a 24-h light conditions. (JPEG 1041 kb)


  1. Ahsan N, Lee DG, Lee SH, Kang KY, Bahk JD, Choi MS, Lee IJ, Renaut J, Lee BH (2007) A comparative proteomic analysis of tomato leaves in response to waterlogging stress. Physiol Plant 131(4):555–570PubMedCrossRefGoogle Scholar
  2. Ahsan N, Lee DG, Lee KW, Alam I, Lee SH, Bahk JD, Lee BH (2008) Glyphosate-induced oxidative stress in rice leaves revealed by proteomic approach. Plant Physiol Biochem 46(12):1062–1070PubMedCrossRefGoogle Scholar
  3. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399PubMedCrossRefGoogle Scholar
  4. Arbona V, Hossain Z, Lopez-Climent MF, Perez-Clemente RM, Gomez-Cadenas A (2008) Antioxidant enzymatic activity is linked to waterlogging stress tolerance in citrus. Physiol Plant 132(4):452–466PubMedCrossRefGoogle Scholar
  5. Barrero J, Rodriguez P, Quesada V, Ponce P, Ponce M, Micoi J (2006) Both abscisic acid (ABA)-dependent and ABA-independent pathways govern the induction of NCED3, AAO3 and ABA1 in response to salt stress. Plant cell Environ 29(10):2000–2008PubMedCrossRefGoogle Scholar
  6. Bellincampi D, Dipierro N, Salvi G, Cervone F, De Lorenzo G (2000) Extracellular H2O2 induced by oligogalacturonides is not involved in the inhibition of the auxin-regulated rolB gene expression in tobacco leaf explants. Plant Physiol 122(4):1379–1386PubMedCrossRefGoogle Scholar
  7. Berberich T, Sugawara K, Harada M, Kusano T (1995) Molecular cloning, characterization and expression of an elongation factor 1 alpha gene in maize. Plant Mol Biol 29(3):611–615PubMedCrossRefGoogle Scholar
  8. Bradford KJ, Hsiao TC (1982) Stomatal behavior and water relations of waterlogged tomato plants. Plant Physiol 70(5):1508–1513PubMedCrossRefGoogle Scholar
  9. Bright J, Desikan R, Hancock JT, Weir IS, Neill SJ (2006) ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J 45(1):113–122PubMedCrossRefGoogle Scholar
  10. Che P, Lall S, Nettleton D, Howell SH (2006) Gene expression programs during shoot, root, and callus development in Arabidopsis tissue culture. Plant Physiol 141(2):620–637PubMedCrossRefGoogle Scholar
  11. Chung S, Parish RW (2008) Combinatorial interactions of multiple cis-elements regulating the induction of the Arabidopsis XERO2 dehydrin gene by abscisic acid and cold. Plant J 54(1):15–29PubMedCrossRefGoogle Scholar
  12. Cornish K, Zeevaart JA (1988) Phenotypic expression of wild-type tomato and three wilty mutants in relation to abscisic Acid accumulation in roots and leaflets of reciprocal grafts. Plant Physiol 87(1):190–194PubMedCrossRefGoogle Scholar
  13. Desikan R, Griffiths R, Hancock J, Neill S (2002) A new role for an old enzyme: nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana. Proc Natl Acad Sci USA 99(25):16314–16318PubMedCrossRefGoogle Scholar
  14. Dörffling K, Dörffling H, Luck E (2009) Improved frost tolerance and winter hardiness in proline overaccumulating winter wheat mutants obtained by in vitro-selection is associated with increased carbohydrate, soluble protein and abscisic acid (ABA) levels. Euphytica 165(3):545–556CrossRefGoogle Scholar
  15. Else MA, Janowiak F, Atkinson CJ, Jackson MB (2009) Root signals and stomatal closure in relation to photosynthesis, chlorophyll a fluorescence and adventitious rooting of flooded tomato plants. Ann Bot 103(2):313–323PubMedCrossRefGoogle Scholar
  16. Guan L, Scandalios JG (1998) Effects of the plant growth regulator abscisic acid and high osmoticum on the developmental expression of the maize catalase genes. Physiol Plant 104(3):413–422CrossRefGoogle Scholar
  17. Hinz M, Wilson IW, Yang J, Buerstenbinder K, Llewellyn D, Dennis ES, Sauter M, Dolferus R (2010) Arabidopsis RAP2. 2: an ethylene response transcription factor that is important for hypoxia survival. Plant Physiol 153(2):757–772PubMedCrossRefGoogle Scholar
  18. Hong JH, Chung G, Cowan AK (2009) Delayed leaf senescence by exogenous lyso-phosphatidylethanolamine: towards a mechanism of action. Plant Physiol Biochem 47(6):526–534PubMedCrossRefGoogle Scholar
  19. Hossain Z, Lopez-Climent MF, Arbona V, Perez-Clemente RM, Gomez-Cadenas A (2009) Modulation of the antioxidant system in Citrus under waterlogging and subsequent drainage. J Plant Physiol 166(13):1391–1404PubMedCrossRefGoogle Scholar
  20. Irfan M, Hayat S, Hayat Q, Afroz S, Ahmad A (2010) Physiological and biochemical changes in plants under waterlogging. Protoplasma 241(1–4):3–17PubMedCrossRefGoogle Scholar
  21. Irving L, Sheng YB, Woolley D, Matthew C (2007) Physiological effects of waterlogging on two lucerne varieties grown under glasshouse conditions. J Agron Crop Sci 193(5):345–356CrossRefGoogle Scholar
  22. Israelsson M, Siegel RS, Young J, Hashimoto M, Iba K, Schroeder JI (2006) Guard cell ABA and CO2 signaling network updates and Ca2+ sensor priming hypothesis. Curr Opin Plant Biol 9(6):654–663PubMedCrossRefGoogle Scholar
  23. Jannat R, Uraji M, Morofuji M, Islam MM, Bloom RE, Nakamura Y, Mcclung CR, Schroeder JI, Mori IC, Murata Y (2011) Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells. J Plant Physiol 168(16):1919–1926PubMedCrossRefGoogle Scholar
  24. Jiang M, Zhang J (2002) Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves. J Exp Bot 53(379):2401–2410PubMedCrossRefGoogle Scholar
  25. Krishnaswamy S, Verma S, Rahman MH, Kav NNV (2011) Functional characterization of four APETALA2-family genes (RAP2. 6, RAP2. 6L, DREB19 and DREB26) in Arabidopsis. Plant Mol Biol 75(1–2):107–127PubMedCrossRefGoogle Scholar
  26. Kumutha D, Ezhilmathi K, Sairam R, Srivastava G, Deshmukh P, Meena R (2009) Waterlogging induced oxidative stress and antioxidant activity in pigeonpea genotypes. Biol Plant 53(1):75–84CrossRefGoogle Scholar
  27. Lee S, Kang J, Park HJ, Kim MD, Bae MS, Choi H, Kim SY (2010) DREB2C interacts with ABF2, a bZIP protein regulating abscisic acid-responsive gene expression, and its overexpression affects abscisic acid sensitivity. Plant Physiol 153(2):716–727PubMedCrossRefGoogle Scholar
  28. Leul M, Zhou W (1999) Alleviation of waterlogging damage in winter rape by uniconazole application: effects on enzyme activity, lipid peroxidation, and membrane integrity. J Plant Growth Regul 18(1):9–14PubMedCrossRefGoogle Scholar
  29. Leung J, Merlot S, Gosti F, Bertauche N, Blatt MR, Giraudat J (1998) The role of ABI1 in abscisic acid signal transduction: from gene to cell. Symp Soc Exp Biol 51:65–71PubMedGoogle Scholar
  30. Licausi F, Van Dongen JT, Giuntoli B, Novi G, Santaniello A, Geigenberger P, Perata P (2010) HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana. Plant J 62(2):302–315PubMedCrossRefGoogle Scholar
  31. Liu PF, Chang WC, Wang YK, Chang HY, Pan RL (2008) Signaling pathways mediating the suppression of Arabidopsis thaliana Ku gene expression by abscisic acid. Biochim Biophys Acta Gene Regul Mech 1779(3):164–174CrossRefGoogle Scholar
  32. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7(9):405–410PubMedCrossRefGoogle Scholar
  33. Obayashi T, Hayashi S, Saeki M, Ohta H, Kinoshita K (2009) ATTED-II provides coexpressed gene networks for Arabidopsis. Nucleic Acids Res 37(Database issue):D987–D991Google Scholar
  34. Onate-Sanchez L, Anderson JP, Young J, Singh KB (2007) AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense. Plant Physiol 143(1):400–409PubMedCrossRefGoogle Scholar
  35. Sairam RK, Dharmar K, Chinnusamy V, Meena RC (2009) Waterlogging-induced increase in sugar mobilization, fermentation, and related gene expression in the roots of mung bean (Vigna radiata). J Plant Physiol 166(6):602–616PubMedCrossRefGoogle Scholar
  36. Saleh A (2003) Plant AP2/ERF transcription factors. Genetika 35(1):37–50CrossRefGoogle Scholar
  37. Samet JS, Sinclair TR (1980) Leaf senescence and abscisic acid in leaves of field-grown soybean. Plant Physiol 66(6):1164–1168PubMedCrossRefGoogle Scholar
  38. Sang J, Jiang M, Lin F, Xu S, Zhang A, Tan M (2008) Nitric oxide reduces hydrogen peroxide accumulation involved in water stress-induced subcellular anti-oxidant defense in maize plants. J Integr Plant Biol 50(2):231–243PubMedCrossRefGoogle Scholar
  39. Scandalios J (2005) Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med Biol Res 38(7):995–1014PubMedCrossRefGoogle Scholar
  40. Song CP, Agarwal M, Ohta M, Guo Y, Halfter U, Wang P, Zhu JK (2005) Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses. Plant Cell 17(8):2384–2396PubMedCrossRefGoogle Scholar
  41. Strizhov N, Abraham E, krész L, Blickling S, Zilberstein A, Schell J, Koncz C, Szabados L (1997) Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. Plant J 12(3):557–569PubMedCrossRefGoogle Scholar
  42. Stuart JM, Segal E, Koller D, Kim SK (2003) A gene-coexpression network for global discovery of conserved genetic modules. Science 302(5643):249–255PubMedCrossRefGoogle Scholar
  43. Sun F, Liu P, Xu J, Dong H (2010) Mutation in RAP2.6L, a transactivator of the ERF transcription factor family, enhances Arabidopsis resistance to Pseudomonas syringae. Physiol Mol Plant Pathol 74(5–6):295–302CrossRefGoogle Scholar
  44. Wu Y, Sanchez JP, Lopez-Molina L, Himmelbach A, Grill E, Chua NH (2003) The abi1-1 mutation blocks ABA signaling downstream of cADPR action. Plant J 34(3):307–315PubMedCrossRefGoogle Scholar
  45. Xiong L, Zhu JK (2003) Regulation of abscisic acid biosynthesis. Plant Physiol 133(1):29–36PubMedCrossRefGoogle Scholar
  46. Yamaguchi-Shinozaki K, Shinozaki K (2005) Organization of cis-acting regulatory elements in osmotic-and cold-stress-responsive promoters. Trends Plant Sci 10(2):88–94PubMedCrossRefGoogle Scholar
  47. Yin D, Chen S, Chen F, Guan Z, Fang W (2009) Morphological and physiological responses of two chrysanthemum cultivars differing in their tolerance to waterlogging. Environ Exp Bot 67(1):87–93CrossRefGoogle Scholar
  48. Zhang X, Zhang Z, Chen J, Chen Q, Wang XC, Huang R (2005) Expressing TERF1 in tobacco enhances drought tolerance and abscisic acid sensitivity during seedling development. Planta 222(3):494–501PubMedCrossRefGoogle Scholar
  49. Zhang G, Tanakamaru K, Abe J, Morita S (2007) Influence of waterlogging on some anti-oxidative enzymatic activities of two barley genotypes differing in anoxia tolerance. Acta Physiol Plant 29(2):171–176CrossRefGoogle Scholar
  50. Zhang G, Chen M, Li L, Xu Z, Chen X, Guo J, Ma Y (2009) Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. J Exp Bot 60(13):3781–3796PubMedCrossRefGoogle Scholar
  51. Zhang H, Mao X, Wang C, Jing R (2010) Overexpression of a common wheat gene TaSnRK2. 8 enhances tolerance to drought, salt and low temperature in Arabidopsis. PLoS ONE 5(12):e16041PubMedCrossRefGoogle Scholar
  52. Zhang Y, Feng F, He C (2012) Downregulation of ospk1 contributes to oxidative stress and the variations in ABA/GA balance in rice. Plant Mol Biol Rep. doi: 10.1007/s11105-011-0386-2 Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.State Ministry of Education Key Laboratory of Integrated Management of Crop PestsNanjing Agricultural UniversityNanjingChina
  2. 2.Institute of Plant ProtectionJiangsu Academy of Agricultural SciencesNanjingChina

Personalised recommendations