Investigating the effect of chitosan on gene expression, p5cs enzyme activity and proline content in rapeseed (Brassica napus L.) under salt stress

Document Type : Research Paper


1 MSc Student, Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran

2 Assistant Professor, Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran.

3 Associate Professor, Department of Molecular Genetics, Faculty of Science, Shahrekord University, Shahrekord, Iran.


Salinity is one of the most important stresses that reduce the yield of most plants. Plants use different mechanisms in response to environmental stresses. Chitosan and its oligomers are used in plants to resist abiotic stresses such as salinity. In this study, the effect of chitosan and salinity on gene expression and p5cs enzyme activity and proline content in rapeseed was investigated.
Materials and methods
For this purpose, rapeseed plants were treated with sodium chloride solution (0, 50, 100, and 150 mM) and chitosan (0, 5, and 10 mg/L). The experiment was performed as a factorial experiment with a completely randomized design in 3 replications. Treated plants were harvested to measure gene expression, p5cs enzyme activity, and proline content.
With increasing salt concentration, the expression of the delta-1 proline-5 carboxylate synthetase (P5CS) gene, enzyme activity, and proline content increased. In the combined salinity of 100 mM with chitosan 10 mg/L, gene expression, activity, and proline content in rapeseed had the highest amount. The use of chitosan in the salt-containing medium compared to the salinity treatments in the same concentration, caused the P5CS gene to be expressed more, increased enzyme activity, and then more proline was synthesized. Therefore, there is a positive correlation between gene expression, enzyme activity, and the amount of proline produced.
According to the results, chitosan with a concentration of 10 mg / l in salinity treatment, by increasing gene expression and the activity of the enzyme delta-1-proline-5-carboxylate synthetase (P5CS), produced proline, which increases the plant's resistance to salinity stress.


عرب پور رق آبادی زهرا، محمدآبادی محمدرضا، خضری امین (1400) الگوی بیانی ژن p32 در بافت‌های ران، دست، راسته و چربی پشت بره کرمانی. مجله بیوتکنولوژی کشاورزی، 13(4)، 183-200.
محمدآبادی محمدرضا (1399) بیان ژن ESR1 در بز کرکی راینی با استفاده از real time PCR‎. مجله بیوتکنولوژی کشاورزی 12(1)، 192-177.
محمدآبادی محمدرضا (1399) پروفایل بیانیmRNA مختص بافت ژن ESR2 در بز. مجله بیوتکنولوژی کشاورزی 12(4)، 184-169.
محمدآبادی محمدرضا، سفلایی محمد (1399). پروفایل بیانی mRNA مختص بافت ژن BMP15 در بز. مجله بیوتکنولوژی کشاورزی 12(3)، 208-191.
محمدآبادی محمدرضا، شبان جرجندی دیانا، عرب پور رق آبادی زهرا، ابارقی فاطمه، ساسان حسینعلی، بردبار فرهاد (1401) نقش رازیانه بر بیان ژن DLK1 در بافت قلب گوسفند. مجله بیوتکنولوژی کشاورزی، 14(2)، 155-133.
محمدآبادی محمدرضا، کرد محبوبه، نظری محمود (1397) مطالعه بیان ژن لپتین در بافت‌های مختلف گوسفند کرمانی با استفاده از real time PCR. مجله بیوتکنولوژی کشاورزی 10(3)، 122-111.
منصوری گندمانی، امیدی ح، رضایی چرمهینی مر (1395) بررسی کاربرد کیتوزان بر جوانه زنی سویا (.Glycine max L) در شرایط تنش شوری. مجله پژوهشهای بذر ایران. 3(2): 178-171.
مهدوی ب، صفری ح. (1394) اثر کیتوزان بر رشد و برخی ویژگیهای فیزیولوژیک نخود در شرایط تنش شوری. فرآیند و کارکرد گیاهی. 4(12): 127-117.
مهدوی ب، مدرس ثانوی س ع م، آقا علیخانی م، شریفی م (1392) اثر غلظتهای مختلف کیتوزان بر جوانه زنی بذر و آنزیم های آنتی اکسیدانت گلرنگ(.Carthamus tinctorius L) در شرایط تنش کم آبی.  مجله پژوهشهای گیاهی (مجله زیست شناسی ایران). 26(2): 365-352.
یامچی ا، رستگار جزی ف، قبادی س، موسوی ا، کارخانه ای ع ا (1383) بیان فراوان ژن Δ– پرولین- ٥-کربوکسیلات سنتتاز (p5cs)، با هدف افزایش مقاومت به تنش های اسموتیک در گیاه تراریخت توتون(Nicotiana tabacum cv.Xanthi). علوم و فنون کشاورزی و منابع طبیعی 8(4): 39-31.
Ali FF, EL-Shehawi AM, Ibrahim OHM, Abdul-Hafeez EY, Moussa MM, Hassan FAS (2021) A vital role of chitosan nanoparticles in improvisation the drought stress tolerance in Catharanthus roseus (L.) through biochemical and gene expression modulation. Plant Physiol Biochem 161: 166- 175.

Alkahtani MDF, Attia KA, Hafez YM, Khan N (2020) Chlorophyll fluorescence parameters and antioxidant defense system can display salt tolerance of salt acclimated sweet pepper plants treated with chitosan and plant growth promoting rhizobacteria. Agronomy 10(8), 1180.

Alqurashi M, Thomas L, Gehring C, Marondedze C (2017) A microsomal proteomics view of H2O2- and ABA-dependent responses. Proteomics 5(3), 22.

Arab L, Ehsanpour AA (2013) Improvement of some physiological responses of alfalfa (Medicago sativa L.) under in vitro salt stress using triadimefon. Prog Biophys Mol (PBS) 3(1), 31-40.
Arabpour Z, Mohammadabadi M, Khezri A (2021) The expression pattern of p32 gene in femur, humeral muscle, back muscle and back fat tissues of Kermani lambs. Agric Biotechnol J 13 (4), 183-200 (In Persian).Amirjani MR (2011) Effect of salinity stress on growth, sugar content, pigments and enzyme activity of rice. Int J Biol Sci (IJBS) 7(1), 73–81.
Baghi zadeh A, Salarizadeh MR, Abaasi F (2014) Effects of salicylic acid on some physiological and biochemical parameters of Brassica napus L.(Canola) under salt stress. Int J Agric Sci (IARAS) 4(2):, 147-52.
Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant and soil 39(1), 205-7.
Bistgani ZE, Siadat SA, Bakhshandeh A et al. (2017) Interactive effects of drought stress and chitosan application on physiological characteristics and essential oil yield of Thymus daenensis Celak. Crop J 5(5), 407-15.
Channuntapipat C, Wirthensohn M, Ramesh SA et al. (2003) Identification of incompatibility genotypes in almond (Prunus dulcis Mill.) using specific primers based on the introns of the S‐alleles. Plant Breed 122(2), 164-8.

Chun SC, Paramasivan M, Chandrasekaran M (2018) Proline accumulation influenced by osmotic stress in arbuscular mycorrhizal symbiotic plants. Front Microbiol 56,1221.

Durgaprasad KM., Muthukumarsamy M, Panneerselvam R (1996) Changes in protein metabolism induced by NaCl salinity in soybean seedlings. Indian J Plant Physiol 1, 98-101.
Elansary HO, Abdel-Hamid AM, Yessoufou K et al. (2020) Physiological and molecular characterization of water-stressed Chrysanthemum under robinin and chitosan treatment. Acta Physiol Plant 42(3), 1-4.
Filippou P, Bouchagier P, Skotti E, Fotopoulos V (2014) Proline and reactive oxygen/nitrogen species metabolism is involved in the tolerant response of the invasive plant species Ailanthus altissima to drought and salinity. Environ Exp Bot 97, 1-0.
Geng W, Li Z, Hassan MJ, Peng Y (2020) Chitosan regulates metabolic balance, polyamine accumulation, and Na+ transport contributing to salt tolerance in creeping bentgrass. BMC Plant Biol. 20, 506.
Guan YJ, Hu J, Wang XJ, Shao CX (2009) Seed priming with chitosan improves maize germination and seedling growth in relation to physiological changes under low temperature stress. J Zhejiang Univ Sci 10(6), 427-33.
Gunstone FD (2004) Rapeseed and canola oil: production, processing, properties and uses. Blackwell Publishing Ltd. 222 pp.
Goharrizi, KJ, Baghizadeh A, Afroushteh M et al. (2020) Effects of salinity stress on proline content and expression of Δ1-pyrroline-5-carboxylate synthase and vacuolar-type H+ subunit E genes in wheat. Plant Genet Resour 18(5), 334-342.
Hare PD, Cress WA, Van Staden J (2003) A regulatory role for proline metabolism in stimulating Arabidopsis thaliana seed germination. Plant Growth Regul 39(1), 41-50.
Hare PD, Cress WA, Van Staden J (2001) The effects of exogenous proline and proline analogues on in vitro shoot organogenesis in Arabidopsis. Plant Growth Regul 34, 203–207.
Hidangmayum A, Dwivedi P, Katiyar D, Hemantaranjan A (2019) Application of chitosan on plant responses with special reference to abiotic stress. Physiol Mol Biol Plants 25, 313-326.

Jimenez-Gomez CP, Cecilia JA (2020) Chitosan: A natural biopolymer with a wide and varied range of applications. Molecules 25(17), 3981.

Khan NA, Ansari HR, Khan M, Mir R (2002) Effect of phytohormones on growth and yield of Indian mustard. Indian J Plant Physiol 7(1), 75-78.
Kenawy ER, Worley SD, Broughton R (2007) The chemistry and applications of antimicrobial polymers: a state-of-the-art review. Biomacromolecules 8(5), 1359-1384.
Kubala S, Garnczarska M, Wojtyla Łet al. (2015) Deciphering priming-induced improvement of rapeseed (Brassica napus L.) germination through an integrated transcriptomic and proteomic approach. Plant Sci 231, 94-113.
Lehmann S, Funck D, Szabados L, Rentsch D (2010) Proline metabolism and transport in plant development. Amino Acids 39(4), 949-962.
Mahdavi B, Modares Sanavi SAM, Agha Alikhani M, Sharifi M (2013) Effect of chitosan on safflower (Carthamus tinctorius L.) seed germination and antioxidant enzymes activity under water stress Iranian J Plant Biol IJPB 26(3), 352- 365 (In Persian).
Mahdavi B, safari H (2015) Effect of chitosan on growth and some physiological characteristics of Chickpea under salinity stress condition. J Plant proc Func 4 (12), 117-127 (In Persian).
Misra N, Saxena P (2009) Effect of salicylic acid on proline metabolism in lentil grown under salinity stress. Plant Sci 177(3),181-9.
Mohammadabadi MR, Shaban Jorjandy D, Arabpoor Raghabadi Z, Abareghi F, Sasan HA, Bordbar F (2022) The role of fennel on DLK1 gene expression in sheep heart tissue. Agric Biotechnol J 14 (2), 155-170. 
Masoudzadeh SH, Mohammadabadi M, Khezri A, et al. (2020) Effects of diets with different levels of fennel (Foeniculum vulgare) seed powder on DLK1 gene expression in brain, adipose tissue, femur muscle and rumen of Kermani lambs. Small Rumin Res 193, e106276.
Mohammadabadi M (2021) Tissue-specific mRNA expression profile of ESR2 gene in goat. Agric Biotechnol J 12 (4), 167-181 (In Persian).
Mohammadabadi M, Masoudzadeh SH, Khezri A, et al. (2021) Fennel (Foeniculum vulgare) seed powder increases Delta-Like Non-Canonical Notch Ligand 1 gene expression in testis, liver, and humeral muscle tissues of growing lambs. Heliyon 7 (12), e08542.
Mohammadabadi M, Soflaei M (2020) Tissue-specific mRNA expression profile of BMP15 gene in goat. Agric Biotechnol J 12, 191-208 (In Persian).
Mohammadabadi MR (2020) Expression of ESR1 gene in Raini Cashmere goat using Real Time PCR. Agric Biotechnol J 12 (1), 177-192 (In Persian).
Mohammadabadi MR, Kord M, Nazari M (2018) Studying expression of leptin gene in different tissues of Kermani Sheep using Real Time PCR. Agric Biotechnol J 10, 111-122 (In Persian).
Munns R (2002) Comparative physiology of salt and water stress. Plant Cell Environ. 25(2), 239-50.
Orcutt DM, Nilsen ET (2000) The physiology of plants under stress, soil and biotic factors. John Wiley and Sons, New York. 177-235.
Parihar P, Singh S, Singh R, Singh VP, Prasad SM (2015) Effect of salinity stress on plants and its tolerance strategies: a review. Environ Sci Pollut Res 22(6), 4056-4075.
Patel H, Krishnamurthy R (2013) Elicitors in plant tissue culture. J pharmacogn phytochem 2(2), 60-5.
Pérez‐Arellano I, Carmona‐Álvarez F, Martínez AI, et al. (2010) Pyrroline‐5‐carboxylate synthase and proline biosynthesis: From osmo tolerance to rare metabolic disease. Protein Sci 19(3), 372-382.
Pottosin I, Velarde-Buendía A.M, Bos, J, et al. (2014) Cross-talk between reactive oxygen species and polyamines in regulation of ion transport across the plasma membrane: implications for plant adaptive responses. J Exp Bot 65(5), 1271-1283.
Rejeb KB, Abdelly C, Savouré A (2014). How reactive oxygen species and proline face stress together. Plant Physiol Biochem 80, 278-284.
Qureshi MI, Abdin MZ, Ahmad J, Iqbal M (2013) Effect of long-term salinity on cellular antioxidants, compatible solute and fatty acid profile of sweet annie (Artemisia annua L.). Phytochemistry 95, 215-223.
Razavizadeh R, Ehsanpour A (2009) Effects of salt stress on proline content, expression of delta-1-pyrroline-5-carboxylate synthetase, and activities of catalase and ascorbate peroxidase in transgenic tobacco plants. Biol Lett 46(2), 63-75.
Silva-Ortega CO, Ochoa-Alfaro AE, Reyes-Agüero JA, et al. (2008) Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear. Plant Physiol Biochem 46(1)m 82-92.
Saleh L, Plieth C (2013) A9C sensitive Cl−-accumulation in A. Thaliana root cells during salt stress is controlled by internal and external calcium. Plant Signal Behav 28(6), e24259.

Sharma A, Shahzad B, Kumar V, Kaur Kohli S (2019) Phytohormones Regulate Accumulation of Osmolytes Under Abiotic Stress Biomolecules 9(7), 285.

Shahsavari M, Mohammadabadi M, Khezri A, et al. (2021) Correlation between insulin-like growth factor 1 gene expression and fennel (Foeniculum vulgare) seed powder consumption in muscle of sheep. Anim Biotechnol 33, 1-11
Soleimani V, Ahmadi J, Golkari S, Sadeghzadeh B (2015) Expression profiling of PAP3, BZIP, and P5CS genes in soybean under drought stress conditions. Turk J Botany. 39(6), 952-961.
Špoljarević M, Agić D, Lisjak M, Gumze Aet al. (2011) The relationship of proline content and metabolism on the productivity of maize plants. Plant Signal Behav.6(2), 251-257.
Tajik H, Moradi M, Rohani SM, Erfani AMet al. (2008) Preparation of chitosan from brine shrimp (Artemia urmiana) cyst shells and effects of different chemical processing sequences on the physicochemical and functional properties of the product. Molecules 13(6):1263-74.
Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35(4), 753-759.
Wang H, Tang X, Wang H, Shao HB (2015) Proline accumulation and metabolism-related genes expression profiles in Kosteletzkya virginica seedlings under salt stress. Front Plant Sci 6(792): 1-9.
Xue GX, Gao HY, Li PM, Zou Q (2004) Effects of chitosan treatment on physiological and biochemical characteristics in cucumber seedlings under low temperature. Physiol Mol Biol Plants 30(4), 441-448.
Yamchi A, Rastgar Jazii F, Ghobadi C, Mousavi A, et al. (2005) Increasing of tolerance to osmotic stresses in tobacco Nicotiana tabacum cv. xanthi through overexpression of p5cs Gene. J Crop Pro Fun 2005 8 (4): 31-40 (In Persian).
Zayed MA, Elkafafi SH, Zedan MG, SherifaFM (2017) Effect of nano chitosan on growth, physiological and biochemical parameters of Phaseolus vulgaris under salt stress. J plant Prod 8 (5): 577 – 585.
Zhang G, Wang Y, Wu K, Zhang Q, Feng Y, Miao Y, Yan Z (2021) Exogenous application of chitosan alleviate salinity stress in lettuce (Lactuca sativa L.). Horticulturae 7, 342.
Zheng JL, Zhao LY, Shen B, Jiang LH et al. (2016) Effects of salinity on activity and expression of enzymes involved in ionic, osmotic, and antioxidant responses in Eurya emarginata. Acta Physiol Plant 38(3), 70.