Identification and Characterization of Genes Responsible for Drought Tolerance in Rice Mediated by Pseudomonas fluorescens

doi:10.1016/j.rsci.2017.04.005

Abstract

Abstract:

Drought is one of the major abiotic stresses which adversely affect crop plants limiting growth and yield potential. Structural and functional characterization of drought stress-induced genes has contributed to a better understanding of how plants respond and adapt to the drought stress. In the present study, differential display technique was employed to study the gene expression of rice plants at the reproductive stage that were subjected to drought stress by withholding water, Pseudomonas fluorescens strain (Pf1) treated plants subjected for drought stress by withholding water and control (well-watered). Differentially expressed cDNAs of six genes (COX1, PKDP, bZIP1, AP2-EREBP, Hsp20 and COC1) were identified, cloned and sequenced. Real-time qPCR analysis showed that all the six genes were upregulated in drought-stressed plants treated with Pf1. This revealed that the remarkable influence of Pf1 colonization leads to drought tolerance at the reproductive stage. These results showed that high levels of gene expression in plants lacking adequate water can be remarkably influenced by Pf1 colonization, which might be a key element for induced systemic tolerance by microbes.

Key words: rice, drought tolerance, Pseudomonas fluorescens, differential display reverse transcription polymerase chain reaction, quantitative real-time PCR, transcript derived fragment

Saakre Manjesh, Meera Baburao Thirthikar, Puthenpeedikal Salim Abida, Mary Ffancies Rose, Poothecty Achuthan Valasala, Thomas George, Radha Sivarajan Sajeevan. Identification and Characterization of Genes Responsible for Drought Tolerance in Rice Mediated by Pseudomonas fluorescens[J]. Rice Science, 2017, 24(5): 291-298.

Figures/Tables 6

References 20

[1]	Auffhammer M, Ramanathan V, Vincent J R.2011. Climate change, the monsoon, and rice yield in India.Clim Change, 111(2): 411-424.
[2]	Ahmad M, Kibert M.2013. Mechanisms and applications of plant growth promoting rhizobacteria: Current perspective. J King Saud Univ Sci, 26(1): 1-20.
[3]	Arshad M, Saleem M, Hussain S.2007. Perspectives of bacterial ACC deaminase in phytoremediation.Trends Biotechnol, 25(8): 356-362.
[4]	Bononi A, Agnoletto C, de Marchi E, Marchi S, Patergnani S, Bonora M, Giorgi C, Missiroli S, Poletti F, Rimessi A, Pinton P. 2011. Protein kinases and phosphatases in the control of cell fate.Enzyme Res, 2011: 1-26.
[5]	Chen L H, Han J P, Deng X M, Tan S L, Li L L, Li L, Zhou J F, Peng H, Yang G X, He G Y, Zhang W X.2016. Expansion and stress responses of AP2/EREBP superfamily in Brachypodium distachyon.Sci Rep, 6: 21623.
[6]	Deveau A, Palin B, Delaruelle C, Peter M, Kohler A, Pierrat J C, Pierrart J C, Sarniguet A, Garbaye J, Martin F, Frey-Klett P.2007. The mycorrhiza helper Pseudomonas fluorescens BBc6R8 has a specific priming effect on the growth, morphology and gene expression of the ectomycorrhizal fungus Laccaria bicolor S238N.New Phytol, 175(4): 743-755.
[7]	Dominguez-Nunez J A, Munoz D, de la Cruz A, Saiz de Omenaca J A.2013. Effects of Pseudomonas fluorescens on the water parameters of mycorrhizal and non-mycorrhizal seedlings of Pinus halepensis.Agron, 3(3): 571-582.
[8]	Ganeshan G, Arthikala A M.2005. Pseudomonas fluorescens, a potential bacterial antagonist to control plant diseases.J Plant Interact, 1(3): 123-134.
[9]	Ghadirnezhad R, Fallah A.2014. Temperature effect on yield and yield components of different rice cultivars in flowering stage.Int J Agron, 2014: 4.
[10]	Halliwell B.2006. Reactive species and antioxidants: Redox biology is a fundamental theme of aerobic life.Plant Physiol, 141(2): 312-322.
[11]	Heinonsalo J, Hurme K R, Sen R.2004. Recent 14C-labelled assimilate allocation to Scots pine seedling root and mycorrhizosphere compartments developed on reconstructed podsol humus, E- and B-mineral horizons.Plant Soil, 259: 111-121.
[12]	Herman M A B, Nault B A, Smart C D.2008. Effects of plant growth promoting rhizobacteria on bell pepper production and green peach aphid infestations in New York.Crop Prot, 27(6): 996-1002.
[13]	IRRI (International Rice Research Institute). 1996. Standard Evaluation System for Rice. Los Banos, the Philippines: International Rice Research Institute: 102.
[14]	Lugtenbergc B, Kamilova F.2009. Plant-growth-promoting rhizobacteria.Annu Rev Microbiol, 63: 541-556.
[15]	Matthijs S, Tehrani K A, Laus G, Jackson R W, Cooper R W, Cornelis P.2007. Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-pythium activity.Environ Microbiol, 9(2): 425-434.
[16]	Riechmann J L, Meyerowitz E M.1998. The AP2/EREBP family of plant transcription factors.Biol Chem, 379(6): 633-646.
[17]	Scheeff E D, Bourne P E.2005. Structural evolution of the protein kinase-like superfamily.PLoS Comput Biol, 1(5): 351-381.
[18]	Singh K B, Foley R C, Onate-Sanchez L.2002. Transcription factors in plant defense and stress responses.Curr Opin Plant Biol, 5(5): 430-436.
[19]	Vleesschauwer D D, Djavaheri M, Bakker P A H M, Hofte M.2008. Pseudomonas fluorescens WCS374r-induced systemic resistance in rice against Magnaporthe oryzae based on pseudobactin-mediated priming for a salicylic acid-repressible multifaceted defense response.Plant Physiol, 148: 1996-2012.
[20]	Yan S P, Tang Z C, Su W A, Sun W N.2005. Proteomic analysis of salt stress responsive proteins in rice root.Proteomics, 5(1): 235-244.

Gene	Sequence (5′-3′)	Fragment length (bp)
COX1	F: CTCCTAGTCGGCCTGATTTC	108
COX1	R: CATGAGCAGTAGCATCCTTGA	108
PKDP	F: CGTTGATAGTCGCCGCTAAA	109
PKDP	R: TTTAAGAGGCGGGAATGGTG	109
bZIP1	F: GAGCGTACTCTGTCCCATTTAG	115
bZIP1	R: GTTCCAGCGATGAGGTTGT	115
AP2- EREBP	F: AGGTAAAGCCCGAGCAATTC	101
AP2- EREBP	R: GCATCGGTGAATGGTGGTATAA	101
Hsp20	F: TGTGTGTCACCACGCTTTA	119
Hsp20	R: CCTCGCATAGACCCATTCATC	119
COC1	F: CACCTCATGACGATGCAAGA	101
COC1	R: GAGCTTGCTCACTCCTTCAA	101

Gene	Sequence (5′-3′)	Fragment length (bp)
COX1	F: CTCCTAGTCGGCCTGATTTC	108
COX1	R: CATGAGCAGTAGCATCCTTGA	108
PKDP	F: CGTTGATAGTCGCCGCTAAA	109
PKDP	R: TTTAAGAGGCGGGAATGGTG	109
bZIP1	F: GAGCGTACTCTGTCCCATTTAG	115
bZIP1	R: GTTCCAGCGATGAGGTTGT	115
AP2- EREBP	F: AGGTAAAGCCCGAGCAATTC	101
AP2- EREBP	R: GCATCGGTGAATGGTGGTATAA	101
Hsp20	F: TGTGTGTCACCACGCTTTA	119
Hsp20	R: CCTCGCATAGACCCATTCATC	119
COC1	F: CACCTCATGACGATGCAAGA	101
COC1	R: GAGCTTGCTCACTCCTTCAA	101

Treatment	Shoot length (cm)	Root length (cm)	Fresh weight (g)	Dry weight (g)	Yield per panicle (g)	1000-grain weight (g)
Control	111.50	26.66	45.620	29.500	3.196	24.520
Water stressed	91.38	15.54	27.420	11.800	1.482	18.700
Water stressed + Pf1-treated	98.86	24.00	34.940	14.400	1.994	21.220
CD (1%)	11.59	3.04	10.248	12.584	0.297	1.747

Treatment	Shoot length (cm)	Root length (cm)	Fresh weight (g)	Dry weight (g)	Yield per panicle (g)	1000-grain weight (g)
Control	111.50	26.66	45.620	29.500	3.196	24.520
Water stressed	91.38	15.54	27.420	11.800	1.482	18.700
Water stressed + Pf1-treated	98.86	24.00	34.940	14.400	1.994	21.220
CD (1%)	11.59	3.04	10.248	12.584	0.297	1.747

Sequence	Gene code	Gene description	Length (bp)	Hit score	E-value	Accession ID	Database
Sequence 1	COX1	Cytochrome c oxidase subunit 1	159	150	0.00	Os12g0561000	IGR, rice
Sequence 2	PKDP	Protein kinase domain protein	161	129	0.00	Os08g39170	IGR, rice
Sequence 3	bZIP1	bZIP family protein	680	83	2e-17	CT833525	PTFD
Sequence 4	AP2-EREBP	APETALA2-ethylene responsive element binding protein	276	79	0.00	OsIBCD031146	DRTF
Sequence 5	Hsp20	Heat shock protein 20	484	230	4e-25	Os01g04380.1	RGAP
Sequence 6	COC1	Circadian oscillator component 1	228	97	3e-06	AY885936	PTFD
IGR, Institute of Genome Research; PTFD, Plant Transcription Factor Database; DRTF, Database of Rice Transcription Factor; RGAP, Rice Genome Annotation Project.