Identification and study of changes in expression pattern of some genes involved in drought tolerance in Lentil (Lens culinaris)

Document Type : Research Paper

Authors

1 M.Sc. Graduate of Genetic and plant breeding, Department of agronomy and plant breeding, Faculty of Sciences and Agricultural Engineering, Tehran University

2 , Department of Agronomy & Plant Breeding, Faculty of Agronomy Sciences, College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran.

3 Associate Professor, Department of Agronomy & Plant breeding, Faculty of Agronomy Sciences, College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran

4 Assistant Professor, Department of Agricultural Biotechnology, Tarbiat Modares University, Tehran, Iran

5 PhD of plant breeding, Department of Agronomy & Plant breeding, Faculty of Agronomy Sciences, College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran

Abstract

Objectives
The research was conducted to study changes in expression pattern of some genes involved in drought tolerance due to the problem of terminal drought stress in rainfed lentil agriculture in Iran.
Material and methods
In order to identify the consensus sequence of the coding sequence (CDS) of selected lentil genes; Aquaporin PIP2, Fructose bisphosphate aldolase, Oxygen-evolving enhancer protein2, WRKY1 and Xyloglucan endotransglucosylase/hydrolase, expressed sequence tag (EST) and reads were identified through similar ortholog sequences in other leguminosae plants. 90, 60 and 30 percent of field capacity were considered as control, medium and severe stress respectively. RNA was extracted from both control and severe drought stress treated plants leaf samples, cDNA was synthesized from RNA samples. Expression profiles of the genes, were determined via real time PCR technique. To represent the effect of drought stress on lentil plant, some morphological, physiological and biochemical characteristics were measured.
Results
Characteristics like plant height, shoot fresh weight, root fresh weight, shoot dry weight, root dry weight, relative water content, leaf area index, chlorophyll and protein content decreased due to drought stress, leaf trichome density, electrolyte leakage index, proline and carbohydrate content, malondialdehyde and hydrogen peroxide increased in drought stress. Analysis of real time PCR demonstrated expression of AQUAPIP2، FBA and WRKY1 genes were increased and expression of XTH and OEE2 genes were decreased under severe drought stress condition.
Conclusions
The results show that AQUAPIP2, FBA and WRKY1 genes are involved in drought tolerance. Due to higher expression of AQUAPIP2 and WRKY1 genes in the tolerant genotype, reduction of RWC in this genotype is less. Less increase in hydrogen peroxide in the tolerant genotype, partly influenced by higher expression of AquaPIP2 gene and higher activity of antioxidant enzymes, which was influenced by higher expression of AquaPIP2 gene in tolerant genotype than susceptible genotype. The increase in carbohydrate content in both genotypes was partially affected by FBA gene expression.

Keywords


علیزاده بهزاد، مصطفوی خدا داد، زمانیان محمد (1395) بررسی میزان تحمل به خشکی در ارقام شبدر برسیم و ایرانی. مجله زراعت و اصلاح نباتات 12، 76-67.
References
Abdullah-Al-Rahad M, Rahman MS, Akter T et al. (2018) Varietal screening of mung bean against whitefly and aphid. J Biosci Agric Res 18, 1478-1487.
Abebe T, Skadsen RW, Kaeppler HF (2004) Cloning and identification of highly expressed genes in barleylemma and palea. Crop Sci 44, 942-950.
Abid G, Muhovski Y, Mingeot D et al. (2017) Identification and characterization of two faba bean (Vicia faba L.) WRKY transcription factors and their expression analysis during salt and drought stress. J Agric Sci 155, 791.
Abraham R (2015) Lentil (Lens culinaris Medikus) Current status and future prospect of production in Ethiopia. Adv Plant Agric Res 2, 45-53.
Aebi HE (1983) Catalase. Meth Enzym Anal 8, 273- 286.
Albouchi A, Béjaoui Z, El Aouni MH (2003) Influence d ‘un stress hydrique modéré ou sévère sur la croissance de jeunes plants de Casuarina glauca Sieb. Colloq congr sci chang. Planét Sécher 14, 137-142.‏
Alexandersson E, Fraysse L, Sjövall-Larsen S et al. (2005) Whole gene family expression and drought stress regulation of aquaporins. Plant Mol Biol 59, 469-484.‏
Alizadeh B, Mostafavi K, Zamanian M (2017) Study of Drought Tolerance of Berseem and Persian Cultivars of Clover. J Agr Plant Breed 12(4), 67-76 (In Persian). 
Antosiewicz DM, Purugganan MM, Polisensky DH et al. (1997) Cellular localization of Arabidopsis xyloglucan endotransglycosylase-related proteins during development and after wind stimulation. Plant Physiol 115, 1319-1328.
Antosiewicz DM, Purugganan MM, Polisensky DH et al. (1997) Cellular localization of Arabidopsis xyloglucan endotransglycosylase-related proteins during development and after wind stimulation. Plant Physiol 115, 1319-1328.
Ashraf M, Mehmood S (1990) Response of four Brassica species to drought stress. Environ Exp Bot 1, 93-100.
Bates LS, Waldern RP, Tear ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39, 205-207.                             
Biju S, Fuentes S, Gupta D (2018) The use of infrared thermal imaging as a non-destructive screening tool for identifying drought-tolerant lentil genotypes. Plant Physiol Biochem 127, 11-24.
Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress. Ann Bot 91, 179-194.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.‏
Cevik S, Akpinar G, Yildizli A et al. (2019) Comparative physiological and leaf proteome analysis between drought-tolerant chickpea Cicer reticulatum and drought-sensitive chickpea C. arietinum. J Biosci 44, 1-13.
Chance B, Maehly AC (1955) Assay of catalases and peroxidases.‏ Meth Enzymol 2, 764-775.
Chiatante D, Di Iorio A, Sciandra S et al. (2006) Effect of drought and fire on root development in Quercus pubescens Willd. and Fraxinus ornus L. seedlings. Environ Exp Bot 56, 190-197.‏
Fan W, Zhang Z, Zhang Y (2009) Cloning and molecular characterization of fructose-1,6-bisphosphate aldolase gene regulated by high-salinity and drought in Sesuvium portulacastrum. Plant Cell Rep 28, 975–984.
FAOSTAT (2008) Available at: http://faostat.fao.org/.
FAOSTAT (2016-2019) Available at: http://faostat.fao.org/.
Feng R, Zhang Y, Yu W et al. (2013) Analysis of the relationship between the spectral characteristics of maize canopy and leaf area index under drought stress. Acta Ecol Sin 33, 301-307.
Flechner A, Gross W, Martin WF,  Schnarrenberger  C (1999)  Chloroplast class I and class II aldolases are bifunctional for fructose‐1,6‐biphosphate and sedoheptulose‐1,7‐biphosphate cleavage in the Calvin cycle. FEBS Lett 447, 200-202.‏
Fracasso A, Trindade L, Amaducci S (2016) Drought tolerance strategies highlighted by two Sorghum bicolor races in a dry-down experiment. J Plant Physiol 190, 1-14.
Fry SC, Smith RC, Renwick KF et al. (1992) Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem 282, 821-828.
Ghanbari AA, Mousavi S H, Pessarakli M (2015) Accumulation of reserve compounds in common bean seeds under drought stress. J Plant Nutr 38, 609-623.
Gopesh GC, Vandemark GJ (2013) Stability of expression of reference genes among different lentil (Lens culinaris) genotypes subjected to cold stress, white mold disease, and Aphanomyces root rot. Plant Mol Biol Rep 31, 1109-1115.
Gunes A, Inal A, Adak MS et al. (2008) Effect of drought stress implemented at pre-or post-anthesis stage on some physiological parameters as screening criteria in chickpea cultivars. Russ J Plant Physiol 55, 59-67.
Guo YY, Yu HY, Yang MM et al. (2018) Effect of drought stress on lipid peroxidation, osmotic adjustment and antioxidant enzyme activity of leaves and roots of Lycium ruthenicum Murr. Seedeling. Russ J Plant Physiol 65, 244–250.
Haake V, Zrenner R, Sonnewald U,  Stitt M (1998) A moderate decrease of plastid aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and inhibits growth of potato plants. Plant J 14, 147-157.
Hajheidari M, Abdollahian‐Noghabi M, Askari H et al. (2005) Proteome analysis of sugar beet leaves under drought stress. Proteomics 5, 950-960.
Harlan JR (1992) Crops and Man. CSSA16, 63-262.
Hazen SP, Pathan MS, Sanchez A et al. (2005) Expression profiling of rice segregating for drought tolerance QTLs using a rice genome array. Funct Integr Genom 5, 104-116.
Heide H, Kalisz HM, Follmann H (2004) The oxygen evolving enhancer protein1 (OEE) of photosystem II in green algae exhibits thioredoxin activity. J Plant Physiol 161, 139-149.‏
Henkes S, Sonnewald U, Badur R et al. (2001) A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. Plant Cell 13, 535-551.
Henkes S, Sonnewald U, Badur R et al. (2001) A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. The Plant Cell 13, 535-551.
Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207, 604-611.
IBPGR, 1985. IBPGR/ ICARDA Lentil Descriptors. International Board For Plant Genetic Resources (IBPGR), AGPG 85/117, Rome, Italy.
Idrissi O, Udupa SM, De Keyser E et al. (2016) Identification of quantitative trait loci controlling root and shoot traits associated with drought tolerance in a lentil (Lens culinaris Medik.) recombinant inbred line population. Front Plant Sci 7, 1174.
Kapanigowda MH, Perumal R, Djanaguiraman M et al. (2013) Genotypic variation in sorghum [Sorghum bicolor (L.) Moench] exotic germplasm collections for drought and disease tolerance. Springer Plus 2, 1-13.
Khan MA, Alghamdi SA, Ammar MH et al. (2019). Transcriptome profiling of faba bean (Vicia faba L.) drought-tolerant variety hassawi-2 under drought stress using RNA sequencing. Electron J Biotechnol 39, 15-29.
Kim EY, Choi YH, Lee JI et al. (2015) Antioxidant activity of oxygen evolving enhancer protein1 purified from Capsosiphon fulvescens. J Food Sci 80, 1412-1417.‏
Koca H, Bor M, Özdemir F et al. (2007) The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environ Exp Bot 60, 344-351.
Kramer PJ (1969) Plant and Soil water Relationships: A Modern synthesis. McGraw-Hill, Inc. New York.
Kumar A, Elston J (1992) Genotypic differences in leaf water relations between Brassica juncea and B. napus. Ann Bot 70, 3-9.‏
Lata C, Jha S, Dixit V et al. (2011) Differential antioxidative responses to dehydration-induced oxidative stress in core set of foxtail millet cultivars [Setaria italica (L.)]. Protoplasma 248, 817-828.‏
Lebherz HG, Leadbetter MM, Bradshaw RA (1984) Isolation and characterization of the cytosolic and chloroplast forms of spinach leaf fructose diphosphate aldolase. Int J Biol Chem 259, 1011-1017.
Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans 11, 591–592.
Livak K J, Schmittgen T D (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25, 402-408.
Lu W, Tang X, Huo Y et al. (2012) Identification and characterization of fructose 1,6-bisphosphate aldolase genes in Arabidopsis reveal a gene family with diverse responses to abiotic stresses. Gene 503, 65-74.
Mensah JK, Obadoni BO, Eruotor PG, Onome-Irieguna, F (2006) Simulated flooding and drought effects on germination, growth, and yield parameters of sesame (Sesamum indicum L.). Afr J Biotechnol 5, 1249-1253.
Mishra BK, Srivastava JP, Lal JP (2018)  Drought resistance in lentil (Lens culinaris Medik.) in relation to morphological, physiological parameters and phenological developments. Int J Curr  Microbiol App Sci 7, 2288-2304.
Mishra BK, Srivastava JP, Lal JP, Sheshshayee MS (2016) Physiological and biochemical adaptations in lentil genotypes under drought stress. Russ J Plant Physiol 63, 695-708.
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7, 405-410.‏
Nadeem M, Li J, Yahya M et al. (2019) Research progress and perspective on drought stress in legumes.  Int J Mol Sci 20, 2541.
Nakano Y, Asada K (1987) Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol 28, 131-140.‏
Niknam V, Razavi N, Ebrahimzadeh H,  Sharifizadeh B (2006) Effect of NaCl on biomass, protein and proline contents, and antioxidant enzymes in seedlings and calli of two Trigonella species. Biol Plant 50, 591-596.
Nishitani K, Tominaga R (1992) Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule. Int J Biol Chem 267, 21058-21064.
Obiadalla-Ali H, Fernie AR, Lytovchenko A et al. (2004) Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size, but only small changes in carbohydrate metabolism. Planta 219, 533-540.
Obiadalla-Ali H, Fernie AR, Lytovchenko A et al. (2004) Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size, but only small changes in carbohydrate metabolism. Planta 219, 533-540.
Öktem HA, Eyidoðan F, Demirba D et al. (2008) Antioxidant responses of lentil to cold and drought stress. J Plant Biochem Biotechnol 17, 15-21.
Papageorgiou GC, Murata N (1995) The unusually strong stabilizing effects of glycine betaine on the structure and function of the oxygen-evolving photosystem II complex. Photosynth Res 44, 243-252.
Pritchard J, Hetherington PR, Fry SC et al. (1993) Xyloglucan endotransglycosylase activity, microfibril orientation and the profiles of cell wall properties along growing regions of maize roots. J Exp Bot 44, 1281-1289.
Purev M, Kim MK, Samdan N,  Yang DC (2008) Isolation of a novel fructose-1, 6-bisphosphate aldolase gene from Codonopsis lanceolata and analysis of the response of this gene to abiotic stresses. Mol Biol 42, 179-186.‏
Ritchie SW, Nguyen HT, Holaday A S (1990) Leaf water content and gas‐exchange parameters of two wheat genotypes differing in drought resistance. Crop Sci 30, 105-111.‏
Sachdeva S, Bharadwaj C, Singh RK et al. (2020) Characterization of ASR gene and its role in drought tolerance in chickpea (Cicer arietinum L.). PloS one 15.
Saeidi K, Zare N, Baghizadeh A, Asghari-Zakaria R (2019) Phaseolus vulgaris genome possesses CAMTA genes, and phavu CAMTA1contributes to the drought tolerance. J Genet 98, 1-14.
Sarker A, Erskine W (2006) Recent progress in the ancient lentil. J Agric Sci 144, 19-29.‏
Shao HB, Chu LY, Jaleel CA, Zhao CX )2008( Water deficit stress induced anatomical changes in higher plants. CR Biol 331, 215-225.
Simova-Stoilova L, Demirevska K, Petrova T et al. (2008) Antioxidative protection in wheat varieties under severe recoverable drought at seedling stage. Plant Soil Environ 54, 529-36.
Singh D, Laxmi A (2015) Transcriptional regulation of drought response: a tortuous network of transcriptional factors. Front Plant Sci 6, 895.
Singh D, Singh CK, Taunk J et al. (2017) Transcriptome analysis of lentil (Lens culinaris Medikus) in response to seedling drought stress. BMC Genom 18, 206.
Sinha R, Pal AK, Singh AK (2018) Physiological, biochemical and molecular responses of lentil (Lens culinaris Medik.) genotypes under drought stress. Indian J Plant Physiol 23, 772-784.
Sonnewald U, Lerchl J, Zrenner R, Frommer W (1994) Manipulation of sink‐source relations in transgenic plants. Plant Cell Environ 17, 649-658.
Sturgeon RJ (1990) Monosaccharides. In Methods in plant biochemistry 2, 1-37.
Talukdar D (2013) Comparative morpho-physiological and biochemical responses of lentil and grass pea genotypes under water stress. J Nat Sci Biol Med 4, 396-402.
Thavarajah D, Thavarajah P, Sarker A et al. (2011) A global survey of effects of genotype and environment on selenium concentration in lentils (Lens culinaris L.): Implications for nutritional fortification strategies. Food Chem 125, 72–76.
Thompson EJ, Smith CR, Fry CS (1997) Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: Evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate. Biochem 327, 699-708.
Tian X, Li S, Liu Y, Liu X (2016) Transcriptomic profiling reveals metabolic and regulatory pathways in the desiccation tolerance of Mungbean (Vigna radiata [L.] R. Wilczek). Front Plant Sci 7, 1921.
Tyerman SD, Niemietz CM, Bramley H (2002) Plant aquaporins: Multifunctional water and solute channels with expanding roles. Plant Cell Environ 25, 173-194.‏
Uozu S, Tanaka-Ueguchi M, Kitano et al. (2000) Characterization of XET-related genes of rice. Plant Physiol 122, 853-860.
Valentovic P, Luxova M, Kolarovic L,  Gasparikova O (2006) Effect of osmotic stress on compatible solutes content, membrane stability and water relations in two maize cultivars. Plant Soil Environ 52, 184.
Velikova V, Yordanov I, Edreva A (2000) Oxidative stress and some antioxidant systems in acid rain-treated bean plants: Protective role of exogenous polyamines. Plant Sci J 151, 59-66.
Vieira CCJ (1991) Flutuaçåes sazonais e caracterizacão parcial dos carboidratos solúveis do órgão subterrâneo de Gomphrena officinalis Mart (Amaranthaceae). MS Thesis, University of São Paulo.
Volkmar KM (1997) Water stressed nodal roots of wheat: Effects on leaf growth. Funct Plant Biol 24, 49-56.
Wang C, Wang L, Yang C, Wang Y (2017) Identification, phylogeny, and transcript profiling of aquaporin genes in response to abiotic stress in Tamarix hispida. Tree Genet Genomes 13, 1-14.
Yang Z, Chi X, Guo F et al. (2020) SbWRKY30 enhances the drought tolerance of plants and regulates a drought stress-responsive gene, SbRD19, in sorghum. J Plant Physiol 246, 153142.‏
Zadražnik T, Moen A, Šuštar-Vozlič J (2019) Chloroplast proteins involved in drought stress response in selected cultivars of common bean (Phaseolus vulgaris L.). 3 Biotech 9, 1-15.
Zeng Y, Tan X, Wang B et al. (2013) Sequence analysis and expression of one fructose-bisphosphate aldolase gene in Camellia oleifera. J Green Sci Technol 1, 30-36.
Zeng Y, Tan X, Zhang L, Jiang N et al. (2014) Identification and expression of fructose-1, 6-bisphosphate aldolase genes and their relations to oil content in developing seeds of tea oil tree (Camellia oleifera). PloS one 9(9), e107422.
Zhang G, Liu Y, Ni Y et al. (2014) Exogenous calcium alleviates low night temperature stress on the photosynthetic apparatus of tomato leaves. PloS one 9(5), e97322.
Zhou S, Hu W, Deng X at al. (2012) Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco. PloS one 7(12), e52439.
Zhuang L, Liu M, Yuan X et al. (2015) Physiological effects of aquaporin in regulating drought tolerance through overexpressing of Festuca arundinacea aquaporin gene FaPIP2. J Am Soc Hortic Sci 140, 404-412.