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



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)


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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

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