DOI: 10.32615/bp.2019.046 BIOLOGIA PLANTARUM 63: 511-518, 2019 This is an open access article distributed under the terms of the Creative Commons BY-NC-ND Licence Exogenous spermidine enhances expression of Calvin cycle genes and photosynthetic efficiency in sweet sorghum seedlings under salt stress A.I. EL SAYED1*, M.A.M. EL-HAMAHMY2, M.S. RAFUDEEN3, and M.K.H. EBRAHIM4,5 Biochemistry Department, Faculty of Agriculture, Zagazig University, 44519 Zagazig, Egypt1 Department of Agricultural Botany, Faculty of Agriculture, Suez Canal University, 41522 Ismailia, Egypt2 Department of Molecular and Cell Biology, University of Cape Town, Private Bag, 7701 Rondebosch, South Africa3 Biology Department, Faculty of applied sciences, Umm Al- Qura University, postal code? Makkah Al- Mukarramah, KSA4 Botany Department, Faculty of Science, Tanta University, 31257 Tanta, Egypt 5 Abstract Salinity adversely affects plants resulting in disruption to plant growth and physiology. Previously, it has been shown that these negative effects can be alleviated by various exogenous polyamines. However, the role of spermidine (Spd) in conferring salinity tolerance in sorghum is not well documented. The effect of exogenous Spd on the responses of sweet sorghum (Sorghum bicolor L.) seedlings to salt stress (150 mM NaCl) was investigated by measuring photosynthetic carbon assimilation, Calvin cycle enzyme activities, and the the expression of respective genes. Application of 0.25 mM Spd alleviated the negative effects of salt stress on efficiency of photosystem II and CO2 assimilation and increased the activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and aldolase. Salt stress significantly lowered the transcriptions of genes encoding Rubisco large subunit, Rubisco small subunit, 3-phosphoglyceric acid kinase, glyceraldehyde-3-phosphate dehydrogenase, triose-3-phosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose- 1,6-bisphosphate phosphatase, and sedoheptulose-1,7-bisphosphatase. However, transcriptions of genes encoding phosphoribokinase and Rubisco were up-regulated. The Spd application enhanced expressions of most of these genes. It appears Spd conferred salinity tolerance to sweet sorghum seedlings by enhancing photosynthetic efficiency through regulation of gene expressions and activities of key CO2 assimilation enzymes. Additional key words: aldolase, CO2 assimilation rate, NaCl, photosystem II, phylogenetic analysis, Rubisco. Introduction protected from these stresses by scavenging free radicals, stabilizing membranes, maintaining a cation-anion balance, Salinity is a major environmental factor inhibiting crop and stimulation of ATP synthesis (Bouchereau et al. 1999, growth and productivity (Parida and Das 2005). It has Ioannidis et al. 2006). Furthermore, polyamines have been observed that in various plants, salinity induces pivotal roles in many other cellular processes including an increase in endogenous polyamines (Das et al. 1995, gene expression, DNA and protein synthesis, regulation Chattopadhayay et al. 2002). Polyamines, such as of ion channels, and providing protection from oxidative putrescine (Put), spermidine (Spd), and spermine (Spm) damage (Duan et al. 2008). Several studies reported are involved in various physiological and biochemical that application of exogenous polyamines is an effective processes related to the regulation of plant responses to approach for enhancing salinity tolerance of plants and different environmental stresses (Takahashi et al. 2010, for protecting plant cell structure; they could be used to Roychoudhury et al. 2011). This allows the plant to be improve the productivity of many crops under salinity Submitted 8 November 2018, last revision 21 January 2019, accepted 29 January 2019. Abbreviations: ΦPS II – photosynthetic quantumyield; Chl - chlorophyll; FBA - fructose-1,6-bisphosphate aldolase also known as aldolase (ALD); FBPase - fructose-l,6-bisphosphate phosphatase; GAPDH - glyceraldehyde-3-phosphate dehydrogenase; MDA - malondialdehyde; PGK - 3-phosphoglyceric acid kinase; PRK - ribulose-5-phosphate kinase; PS - photosystem; Put - putrescine; RbcL - ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit; RbcS - ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit; RCA - ribulose bisphosphate carboxylase/oxygenase activase; Rubisco - ribulose bisphosphate carboxylase/oxygenase; SBPase - sedoheptulose-1,7-bisphosphatase; Spd - spermidine; Spm - spermine; TPI - triose-3-phosphate isomerase. Acknowledgement: The author thanks the Zagazig University for supporting the research. * Corresponding author: e-mail: [email protected] 511 A.I. EL SAYED et al. stress (Chattopadhayay et al. 2002, Verma and Mishra grown in pots containing washed quartz sand with a half 2005, Chai et al. 2010, Roychoudhury et al. 2011, Shu strength Hoagland nutrient solution (three plants per pot) et al. 2014). Spermidine is the most important PA and is in a growth chamber with a 12-h photoperiod, an irradiance closely associated with stress tolerance in several plants of 450 μmol m-2 s-1, day/night temperatures of 25/20 °C, (Kasukabe et al. 2004). It was shown that the application and a 60 % relative humidity. Ten-day-old seedlings were of Spd results in enhancing salinity tolerance of maize subjected to salt stress by adding NaCl to the Hoagland (Jiang et al. 2000) and cucumber (Duan et al. 2008, Shu nutrient solution. To avoid osmotic shock, the seedlings et al. 2014). Our previous study reported that application were exposed to an initial concentration of 50 mM NaCl, of Spd successfully alleviates salt stress by enhanced which was gradually increased by 50 mM a day until the antioxidant defense systems in wheat seedlings (El Sayed desired concentration was reached (100 or 150 mM). We et al. 2018). Furthermore, exogenous Spd plays major roles used five different treatments:1 ) control - the half-strength in preventing plasma membrane damage in rice plants Hoagland solution; 2) the half-strength Hoagland solution in response to salinity stress by preserving membrane with 100 mM NaCl); 3) the half-strength Hoagland integrity (Roy et al. 2005 Roychoudhury et al. 2011). solution with 150 mM NaCl; 4) the half-strength Hoagland This is supported by studies which show Spd-mediated solution with 100 mM NaCl and 0.25 mM Spd; and 5) the protection of antioxidant capacity and decreased content half-strength Hoagland solution with 150 mM NaCl and of superoxide anion and malondialdehyde (Liu et al. 2006, 0.25 mM Spd. The experiments were done in triplicates. Farooq et al. 2009). It is also speculated that Spd could act Samples of fresh seedlings were harvested, directly placed in stress signaling pathways (Kasukabe et al. 2004). into liquid nitrogen, and stored at -80 °C before analyses. Salinity stress induces a decrease in photosynthetic To determine fresh mass (f.m.), seedlings was weighed efficiency that is often associated with inhibition of after washing with sterile double distilled water. Then they photosystem II (PSII) activity (Lu and Vonshak 2002, Xia were dried in an oven at 70 °C for 72 h to determine dry et al. 2004, Kalaji et al. 2011), and it is considered to be mass (d.m.). the main site of salt stress-mediated damage to electron transport processes (Baker 1991, Mehta et al. 2010). Plants Chlorophyll content, PS II quantum yield, and CO2 treated with Spd alleviate the salinity-induced inhibition fixation rate: Leaves of sweet sorghum seedlings were of photosynthetic capacity, but this effect depends on harvested, acetone (80 %, v/v) was used for pigment Spd concentration and the severity of stress (Shu et al. extraction, and chlorophyll (Chl) a and Chl b were 2014). It has been reported that exogenous Spd increases analyzed spectrophotometrically at the 663 and 645 nm, photosynthetic rates and photochemical efficiency of PS II respectively. The relative content of Chl a and Chl b of cucumber seedlings under salt stress (Duan et al. 2008). was calculated according to Lichtenthaler (1987). The Despite studies showing the involvement of polyamines quantum yield of PS II was measured as a variable to in plant responses to abiotic stresses, the mechanism of maximum Chl fluorescence ratio (FV/FM) using a Mini- exogenous Spd alleviation of salt induced inhibition of PAM fluorometer (Walz, Effeltrich, Germany) as described photochemical efficiency in higher plants remains largely by Oelze et al. (2012). Carbon dioxide gas exchange rates unknown. of sweet sorghum leaves under different treatments were In the present study, we tried to understand the measured with a portable gas exchange system (GFS-3000, physiological function of exogenous Spd in the tolerance Walz, Effeltrich, Germany). of sweet sorghum to salt stress. The importance of this crop not only for feeding animals but also as an alternative Rubisco and aldolase activities: Rubisco activity in source of energy has been highlighted (Smith and Buxton leaf samples (200 mg) was estimated following the 1993, Steduto et al. 1997). In addition, various sweet method of Wang et al. (2009) with some modification. sorghum cultivars are able to grow under soil salinity and A homogenization buffer consisted of 33 mM Tris-HCl water deficiency. We therefore examined how exogenous (pH 7.5), 0.67 mM Na2-EDTA, 33 mM MgCl2, and 10 mM 3 Spd affects sweet sorghum response to salinity with NaHCO3. The initial activity was determined in 150 mm respect to photosynthetic capacity, carbon assimilation, of assay mixture containing 100 mM Tris-HCl