Abstract
To improve the salinity tolerance of rice, a DEAD-box helicase gene isolated from pea with a CaMV35S promoter was transformed into the Bangladeshi rice variety Binnatoa through Agrobacterium-mediated transformation. The transgenic seedlings showed significantly higher chlorophyll content, but decreased root length compared to wild type (WT) under normal physiological conditions. Their status was confirmed by polymerase chain reaction (PCR), semi-quantitative reverse-transcription PCR and Southern blot hybridization for positive integration of the transgene. The T2 progenies from three independent transformation events were characterized for salinity tolerance both at seedling and reproductive stages. Compared to the WT plants, the average decrease in chlorophyll content and dry weight of seedling leaves was lower by 20 and 12% respectively at 12 deciSiemens per meter (dS/m) NaCl stress in hydroponics. A higher leaf K+/Na+ ratio of 0.346 was maintained by the transgenic lines compared to the WT ratio of 0.157, which indicated induced ion homeostasis. At the reproductive stage, transgenic rice plants expressing PDH45 showed better fertility and produced higher grain yield by 16% compared to WT plants under continuous stress of 6 dS/m from 30 days till maturity. One of the transformed lines, PDH45-P3, outperformed the others, and replicated data in reproductive stage soil stress of 12 dS/m NaCl showed its enhanced fertility and yield by 46 and 29% over WT, respectively.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bohra JS, Dorffling K (1993) Potassium nutrition of rice (Oryza sativa L.) varieties under NaCl salinity. Plant Soil 152:299–303
Chaudhury AM, Koltunow A, Payne T, Luo M, Tucker MR, Dennis ES, Peacock WJ (2001) Control of early seed development. Annu Rev Cell Dev Biol 17:677–699
Fukuda A, Nakamura A, Tagiri A, Tanaka H, Miyao A, Hirochika H, Tanaka Y (2004) Function, intracellular localization and the importance in salt tolerance of a vacuolar Na(+)/H(+) antiporter from rice. Plant Cell Physiol 45(2):146–159
Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA 99(25):15898–15903
Gregorio G, Senadhira D, Mendoza R (1997) Screening rice for salinity tolerance. International Rice Research Institute, Los Banos. IRRI discussion paper series no. 22
IRRI (1996) Standard evaluation system manual, 4th edn. International Rice Research Institute, Los Banos
Islam S, Seraj Z (2009) Vacuolar Na+/H+ antiporter expression and salt tolerance conferred by actin1D and CaMV35S are similar in transgenic Binnatoa rice. Plant Tissue Cult Biotechnol 19:257–262
Jefferson RA, Kavanagh TA, Bevan MW (1987) Gus fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901–3907
Jiang X, Leidi EO, Pardo JM (2010) How do vacuolar NHX exchangers function in plant salt tolerance? Plant Signal Behav 5(7):792–795
Karim Z, Iqbal A (2001) Impact of land degradation in Bangladesh. Changing scenario in agricultural land use. Compilation of data from the geographical information system (GIS). Bangladesh Agricultural Research Council Publication, Bangladesh
Khanna HK, Raina SK (1999) Agrobacterium-mediated transformation of indica rice cultivars using binary and superbinary vectors. Aust J Plant Physiol 26:311–324
Kronzucker HJ, Britto DT (2011) Sodium transport in plants: a critical review. New Phytol 189(1):54–81
Lee KS, Choi WY, Ko JC, Kim TS, Gregorio GB (2003) Salinity tolerance of japonica and indica rice (Oryza sativa L.) at the seedling stage. Planta 216(6):1043–1046
Li SC, Chung MC, Chen CS (2001) Cloning and characterization of a dead box RNA helicase from the viable seedlings of aged mung bean. Plant Mol Biol 47(6):761–770
Liu HH, Liu J, Fan SL, Song MZ, Han XL, Liu F, Shen FF (2008) Molecular cloning and characterization of a salinity stress-induced gene encoding DEAD-box helicase from the halophyte Apocynum venetum. J Exp Bot 59(3):633–644
Luo Y, Liu YB, Dong YX, Gao XQ, Zhang XS (2009) Expression of a putative alfalfa helicase increases tolerance to abiotic stress in arabidopsis by enhancing the capacities for ROS scavenging and osmotic adjustment. J Plant Physiol 166(4):385–394
Owttrim GW, Hofmann S, Kuhlemeier C (1991) Divergent genes for translation initiation factor eIF-4A are coordinately expressed in tobacco. Nucleic Acids Res 19(20):5491–5496
Owttrim GW, Mandel T, Trachsel H, Thomas AA, Kuhlemeier C (1994) Characterization of the tobacco eIF-4A gene family. Plant Mol Biol 26(6):1747–1757
Pause A, Methot N, Svitkin Y, Merrick WC, Sonenberg N (1994) Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation. EMBO J 13(5):1205–1215
Pham XH, Reddy MK, Ehtesham NZ, Matta B, Tuteja N (2000) A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase 1 activity. Plant J 24(2):219–229
Ponnamperuma F (1977) Screening rice for tolerance to mineral stress. IRRI research paper series no. 6
Rasul N (2005) Isolation and characterization of vacuolar Oryza sativa Na+/H+ antiporter and its expression in transgenic rice. PhD thesis, University of Dhaka
Rasul N, Ali K, Islam R, Seraj ZI (1997) Transformation of an indica rice cultivar Binnatoa with Agrobacterium tumefaciens. Plant Tissue Cult 7:71–80
Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Sanan-Mishra N, Pham XH, Sopory SK, Tuteja N (2005) Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc Natl Acad Sci USA 102(2):509–514
Tuteja N (2003) Plant DNA helicases: the long unwinding road. J Exp Bot 54(391):2201–2214
Tuteja N, Tuteja R (2004) Unraveling DNA helicases. Motif, structure, mechanism and function. Eur J Biochem 271(10):1849–1863
Vernon L (1969) Spectrophotometric determination of chlorophylls and phaeophytins in plant extracts. Anal Chem 32:1144–1150
Wang JC, Caron PR, Kim RA (1990) The role of DNA topoisomerases in recombination and genome stability: a double-edged sword? Cell 62(3):403–406
Yoine M, Nishii T, Nakamura K (2006) Arabidopsis UPF1 RNA helicase for nonsense-mediated mRNA decay is involved in seed size control and is essential for growth. Plant Cell Physiol 47(5):572–580
Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Laboratory manual for physiological studies of rice, 3rd edn. International Rice Research Institute, Los Banos, pp 61–66
Acknowledgments
Funds for this work are gratefully acknowledged from both the US Department of Agriculture and Bangladesh Academy of Sciences (BAS) for consumables and fellowships to MA, SME, AH, AF and MSR. Technical support from Rabin Sarker for the transformation, Shamim Hossain for DNA isolation and Nazrul Islam for maintenance of the plants is acknowledged. The latter personnel were also supported from funds by the USDA and BAS. Narendra Tuteja thanks the Department of Biotechnology, Government of India, for the funding.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
11032_2011_9625_MOESM2_ESM.eps
Supplementary Figure 1: Construction of pCAMBIA1301-PDH45 vector. (A) pRT100 vector containing CaMV35S promoter and Xba1 site. (B) pBI121-PDH45 sense construct. (C) PDH45 from pBI121-PDH45 was cut by Xba1 and inserted into the MCS of pRT100. (D) CaMV35S-PDH45-polyA fragment was cut out with Pst1 and (E) inserted into MCS of pCAMBIA1301 containing hpt and GUS gene (EPS 5709 kb)
11032_2011_9625_MOESM3_ESM.tif
Supplementary Figure 2: Confirmation of transgene integration. (a) PCR of T1 lines with helicase gene specific primers. (b) Transgene expression in PDH45-P3 plant using semi-quantitative RT-PCR (c) Southern hybridization for confirmation of integration of the PDH45 gene (TIFF 21 kb)
11032_2011_9625_MOESM7_ESM.tif
Supplementary Figure 4a Comparison of grain width among WT, PDH45-P3 and BA70 under control and 12 dS/m NaCl stress (TIFF 3779 kb)
11032_2011_9625_MOESM8_ESM.tif
Supplementary Figure 4b: Comparison of panicle length and spikelet fertility among WT, BA70 and PDH45-P3 at 12 dS/m. The white spikelets are empty shells with no grains inside. Greater number of filled spikelets is seen for the PDH45-P3 transgenic line. (TIFF 2148 kb)
11032_2011_9625_MOESM9_ESM.tif
Supplementary Figure 5: K+/Na+ ratio in wild-type and transgenic rice for reproductive stage after NaCl stress at 12 dS/m. Each bar represents the mean ± SE (n = 6 for WT and BA70, n = 15 for PDH45-P3, P < 0.05) (TIFF 63 kb)
Rights and permissions
About this article
Cite this article
Amin, M., Elias, S.M., Hossain, A. et al. Over-expression of a DEAD-box helicase, PDH45, confers both seedling and reproductive stage salinity tolerance to rice (Oryza sativa L.). Mol Breeding 30, 345–354 (2012). https://doi.org/10.1007/s11032-011-9625-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11032-011-9625-3