Journal of Plant Physiology 175 (2015) 9–20

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Physiology

Enhanced drought tolerance in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene via NO and Ca2+ Baoyun Qian a,b , Xia Li a,∗ , Xiaolong Liu a,b , Pingbo Chen a , Chengang Ren a , Chuanchao Dai c a Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Jiangsu High Quality Rice R & D Center, Nanjing Branch, China National Center for Rice Improvement, Provincial Key Laboratory of Agrobiology, Nanjing 210014, PR China b College of Life Science, Nanjing Agricultural University, Nanjing 210095, PR China c Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, PR China

a r t i c l e

i n f o

Article history: Received 20 March 2014 Received in revised form 20 July 2014 Accepted 28 September 2014 Available online 18 November 2014 Keywords: Rice (Oryza sativa L.) Drought Phosphoenolpyruvate carboxylase PEG6000 Nitric oxide Ca2+

a b s t r a c t We determined the effects of endogenous nitric oxide and Ca2+ on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4 pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) under drought. In this study, seedlings were subjected to PEG 6000 treatments using PC and wild type (WT; Kitaake). The results showed that, compared with WT, PC had higher relative water content (RWC) and net photosynthetic rate (Pn) under drought. During a 2-day re-watering treatment, Pn recovered faster in PC than in WT. Further analyses showed that, under the drought treatment, the amount of endogenous hydrogen peroxide (H2 O2 ) increased in WT mainly via NADPH oxidase. While in PC, the endogenous nitric oxide (NO) content increased via nitrate reductase and nitric oxide synthase on day 2 of the drought treatment and day 1 of the re-watering treatment. After 2 days of drought treatment, PC also showed higher PEPC activity, calcium content, phospholipase D (PLD) activity, C4 -pepc and NAC6 transcript levels, and protein kinase activity as compared with PC without treatment. These changes did not occur in WT. Correlation analysis also proved NO associated with these indicators in PC. Based on these results, there was a particular molecular mechanism of drought tolerance in PC. The mechanism is related to the signaling processes via NO and Ca2+ involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4 pepc. Some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance. © 2014 Published by Elsevier GmbH.

Introduction Drought is one of the most important environmental stress factors limiting plant growth and crop yield (Terzi and Kadioglu, 2006). According to the available data, approximately 45% of the world’s

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; DTT, dithiothreitol; DW, dry weight; EDTA, ethylene diamine tetraacetic acid; FW, fresh weight; Gs, stomatal conductance; H2 O2 , hydrogen peroxide; Ci, intercellular CO2 concentration; MDA, malondialdehyde; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NR, nitrate reductase; O2 − • , superoxide anion radicals; • OH, hydroxyl radicals; PA, phosphatidic acid; PEPC, phosphoenolpyruvate carboxylase; PEPC-k, phosphoenolpyruvate carboxylase-kinase; PLD, phospholipase D; PPFD, photosynthetic photon flux density; Pn, net photosynthetic rate; POD, peroxidase; PVP, polyvinyl pyrrolidone; ROS, reactive oxygen species; Rubisco, ribulose-1,5-bisphosphate-carboxylase/oxygenase; RWC, relative water content; SOD, superoxide dismutase; TW, turgid weight; VPD, vapor pressure difference. ∗ Corresponding author. Tel.: +86 2584390361; fax: +86 2584390322. E-mail addresses: [email protected], [email protected] (X. Li). http://dx.doi.org/10.1016/j.jplph.2014.09.019 0176-1617/© 2014 Published by Elsevier GmbH.

agricultural lands are subjected to frequent drought stress, and 38% of the world’s human population resides in these areas (Ashraf and Foolad, 2007). Plants subjected to water stress can be more sensitive than unstressed plants to other biotic and abiotic stresses such as bacterial or fungal pathogens and competition from weeds. Thus, their productivity may be further limited under field conditions (Caruso et al., 2008). Hence, how to address problems associated with drought is an important focus of current research (Wardle, 2013). The photosynthetic responses to drought involve interactions between physical and metabolic mechanisms (Pinheiro and Chaves, 2011). Compared with C3 plants, C4 plants have higher photosynthetic capacity, and higher nitrogen and water use efficiencies under drought conditions (Zhu et al., 2010). Therefore, researchers hope to introduce C4 features into C3 plants to improve their photosynthetic efficiency and yield (von Caemmerer et al., 2012). Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme in higher plants, and it is also widely distributed

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in green algae and bacteria (Sage et al., 2012). In higher plants, there are several isoforms of PEPC with different organ specificities. These enzymes are involved in a variety of functions including stomata opening, fruit ripening, and seed maturation (Chollet et al., 1996). The leaves of C4 and Crassulacean acid metabolism plants contain high levels of PEPC, which catalyzes the initial CO2 fixation during photosynthesis (Fukayama et al., 2003). Previously, transgenic rice lines expressing high levels of the maize C4 -pepc gene (designated as PC lines) were successfully produced through transgenic technology (Ku et al., 1999). To date, several C3 species have been genetically modified to overproduce PEPC (Miyao et al., 2011). Several studies have reported the detrimental effect of increased PEPC activity on Pn or biomass: for example, overproduction of C4 -pepc in rice resulted in reduced photosynthetic rates because of increased respiration under light conditions (Fukayama et al., 2003) and severely stunted growth (Chen et al., 2004). Other studies showed that PEPC-expressing transgenic plants have a relatively high Pn under stress conditions such as photo-oxidation, heat, and drought (Jiao et al., 2002, 2005; Bandyopadhyay et al., 2007; Fang et al., 2008; O’leary et al., 2011; Ling et al., 2014). Previous studies on PC lines showed that hydrogen peroxide (H2 O2 ), calcium ions (Ca2+ ), and nitric oxide (NO) donors play important roles in regulating stomata movement (Li et al., 2011; Ren et al., 2014; Chen et al., 2014). There are conflicting reports about the effects of PEPC overproduction on photosynthesis, with no clear consensus until now (Miyao et al., 2011). The different results of the various studies could be because of differences in the experimental or measurement procedures used, or because of differences in the expression level of the introduced Zmpepc among rice lines. Positional effects may have resulted in different expression levels of the trans-gene, in spite of the use of the constitutive ZmUbi-1 promoter. Although there is some disagreement about the effects of the C4 -pepc trans-gene on photosynthesis, there is a general consensus that C4 -pepc transgene expression positively affects stress tolerance, although little is known about the underlying molecular mechanisms. In this study, analyses of transgenic rice overexpressing maize C4 -pepc (PC) and Kitaake (WT) provided evidence for the molecular mechanism of drought tolerance in PC. In PC, up-regulation of NO and down-regulation Ca2+ induces higher protein kinase activity for up-regulation of PEPC activity, enhances upstream gene expression of the transcription factor, such as NAC6, then induces higher downstream gene expression C4 -pepc, and cu/zn-sod, which encode components of the antioxidant defense system. The increased expression of these genes enhances the photosynthetic capability of the transgenic line under stress conditions. Materials and methods Plant materials and treatments We used the transgenic rice (Oryza sativa L.) line PC, which over-expresses maize pepc. We used tenth generation plants (Jiao et al., 2002), which were derived from third generation plants (Ku et al., 1999). Wild-type (WT) rice plants (cv. Kitaake) were used as the control. The seeds were surface sterilized with 0.1% (w/v) mercuric chloride solution for 15 min, rinsed three times with 75% (v/v) ethyl alcohol, and then rinsed five times with distilled water. The sterilized seeds were germinated in Petri dishes on two layers of wet filter paper in a controlled incubator at 30 ◦ C in darkness. After 4 days, seedlings were transferred to vessels containing 1/4 modified Hoagland solution with the following nutrients: KNO3 , 0.5 mM; Ca(NO3 )2 , 1.0 mM; KH2 PO4 , 1.0 mM; MgSO4 , 0.3 mM; H3 BO3 , 13.3 mM; MnCl2 , 3.0 mM; CuSO4 , 0.5 mM; ZnSO4 , 1.0 mM; Na2 MoO4 , 0.1 mM; NaCl, 2 mM; CoCl2 , 0.01 mM; NiSO4 , 0.1 mM; and Ethylenediamine-N,N9 -bis(2-hydroxyphenylacetic acid) Ferric sodium complex, 20 mM (Jones, 1982). The solution pH was

adjusted to 5.8 (control condition) daily, and the solution was replaced every 2 day. When the seedlings reached the three-leaf stage, they were transferred to vessels containing full-strength modified Hoagland solution (Jones, 1982). The vessels were placed in a controlled growth chamber (14 h light/10 h dark photoperiod; light intensity, 600 ␮mol m−2 s−1 ; 30 ◦ C). When the sixth leaves from the base were fully expanded, they were collected and used for investigations. Plants were subjected to a PEG-6000 treatment to simulate drought stress. The plants were transferred into culture solution containing 15% (w/v) polyethylene glycol (PEG)-6000. Then, after 2 days, the treated materials were transferred to a culture solution without PEG-6000 and were grown for a further 2 days. We evaluated the various parameters every day during the 4-day treatment. Each treatment was replicated three times. The sixth mature leaves from the base were collected at selected time points. The relative water content (RWC) of the fresh leaves was determined immediately. For other assays, leaf samples were frozen in liquid nitrogen (N2 ) and stored at −75 ◦ C until analysis. Measurement of relative water content (RWC) To measure RWC, 10 leaf discs (0.5 cm-diameter) per replicate were obtained from the central third of leaves using a circular cutter. The discs were weighed to obtain fresh weight (FW), and then floated on water at 4 ◦ C in the dark. After 18 h, the turgid weight (TW) was measured. Leaf discs were then dried at 75 ◦ C to constant weight and dry weight (DW) was obtained. The RWC (%) was calculated as follows: RWC = (FW − DW)/(TW − DW) × 100(Anjumet al., 2011).

Measurement of net photosynthetic rate (Pn) Gas exchange was measured with an open gas-exchange system (LI-6400, Li-Cor, Lincoln, NE, USA). Illumination was provided by light-emitting diodes (470 and 665 nm; Li-Cor). The leaf-toair vapor pressure difference (VPD) was controlled using a dew point generator (LI-610; Li-Cor). Measurements of net photosynthetic rate (Pn), stomata conductance (Gs), and intercellular CO2 concentration (Ci) were performed under the following conditions: leaf temperature, 30 ◦ C; 360 ␮mol mol−1 CO2 ; 21% O2 ; photosynthetic photon flux density (PPFD) 800 ␮mol m−2 s−1 ; flow flux, 500 ␮mol s−1 ; VPD, 1.0–1.2 kPa. Before all measurements, the uppermost fully expanded leaf was placed in the leaf chamber and exposed to 500 ␮mol m−2 s−1 PPFD at a leaf temperature of 30 ◦ C in ambient air for 30 min. Each treatment was replicated three to five times (Li et al., 2011). Antioxidant enzyme assays Plant tissue (0.15 g, FW) was homogenized in 1.5 ml extraction buffer (50 mmol L−1 phosphate buffer, 1 mmol L−1 ethylene diamine tetraacetic acid (EDTA), 1% (w/v) polyvinyl pyrrolidone (PVP), pH 7.4) using a mortar and pestle on ice. The homogenate was centrifuged at 12,000 × g for 10 min at 4 ◦ C, and the supernatant was used for assays. Superoxide dismutase (SOD) activity was assayed by measuring the ability of the enzyme in the crude extract to inhibit the photochemical reduction of nitroblue tetrazolium (NTB) by photo-chemically generated superoxide radicals. This reaction was monitored by measuring the change in absorbance at 560 nm using a UV-1200 spectrophotometer (Meipuda, Shanghai, China). One unit (U) of SOD was defined as the amount of enzyme required to inhibit the rate of nitroblue tetrazolium reduction by 50% at 25 ◦ C (Giannopolitis and Ries, 1977). Peroxidase (POD) activity was

B. Qian et al. / Journal of Plant Physiology 175 (2015) 9–20

measured as described by Gao et al. (2008). Briefly, the reaction mixture consisted of 2.8 mL 3% glycerol, 0.1 mL 2% H2 O2 , and 0.1 mL crude enzyme solution. The absorbance of the solution was measured at 470 nm. One unit of enzyme activity was defined as the amount that caused a 0.1 increase in the absorbance value per min. Catalase (CAT) activity was measured as described by Jiang and Zhang (2001). The absorbance of the reaction mixture (200 ␮L 50 mmol L−1 phosphate buffer (pH 7.0), 10 mmol L−1 H2 O2 ) was measured at 240 nm. Ascorbate peroxidase (APX) activity was measured as described by Saruyama and Tanida (1995). The absorbance of the reaction mixture (50 mmol L−1 phosphate buffer solution (pH 7.0), 0.5 mmol L−1 ascorbic acid, 0.1 mmol L−1 EDTA, 0.1 mmol L−1 H2 O2 , and 0.1 mL crude enzyme solution) was measured at 290 nm. One unit of enzyme activity was defined as the amount that caused a 0.1 increase in absorbance per min. The soluble protein content was estimated by the method of Bradford (1976), using bovine serum albumin as the standard. Measurement of superoxide anion radical concentration and malondialdehyde (MDA) content The superoxide anion radical concentration was determined using the method of Elstner and Heupel (1976). Briefly, approximately 1 g leaf tissue was added to 3 mL 65 mmol potassium phosphate buffer (pH 7.8) and mechanically mixed. After centrifugation at 9000 × g for 15 min, the superoxide anion radical concentration in the supernatant was determined. An aliquot (0.15 mL) contained 65 mmol potassium phosphate buffer was added to the following solutions: 0.1 ml 10 mmol hydroxylamine hydrochloride was added to the test group and zero groups (replaced by 0.1 mL d H2 O in the control group). All the aliquots were incubated in a water bath at 25 ◦ C for 10 min. Next, 0.5 mL supernatant was added to the experimental and control groups (0.5 mL of 65 mmol potassium phosphate buffer was added to the blank zero group), and the mixtures were incubated in a water bath at 25 ◦ C for 20 min. Subsequently, 1 mL 58 mmol sulfanilamide and 1 mL 7 mmol ␣-naphthylamine were added to each tube. The tubes were incubated in a water bath at 25 ◦ C for 20 min. Then, an equal volume (3 mL) of chloroform was added and the samples were centrifuged at 10,000 × g for 3 min. The absorbance of the aqueous phase was measured at 530 nm. MDA content was determined using the thiobarbituric method as described by Dhindsa and Matowe (1981). The absorbance of the reaction mixture was measured at 532 nm. Measurement of hydrogen peroxide (H2 O2 ) content Hydrogen peroxide (H2 O2 ) content was determined as described by Patterson et al. (1984) and Ren et al. (2014). Leaf tissue (0.3 g) was homogenized in 3 ml acetone. The homogenate was then centrifuged at 10,000 × g for 10 min at 4 ◦ C, and the supernatant was used for the assay. The supernatant was collected and added to 0.3 mL of a concentrated hydrochloric acid solution (containing 0.1 mL 20% TiCl4 and 0.2 mL concentrated ammonia). The mixture was incubated at 25 ◦ C for 10 min, and then centrifuged at 8000 × g for 10 min at 4 ◦ C. The pellet was washed twice with cold acetone, and then 3 mL 1 mol L−1 H2 SO4 was added. The absorbance of the solution was measured at 410 nm, and the amount of H2 O2 was calculated from a standard curve prepared using known concentrations of H2 O2 . Measurement of calcium ions (Ca2+ ) content The calcium ions (Ca2+ ) content was measured as described by Yang et al. (1998). Leaf tissue (0.1 g) was homogenized in 1 mL extraction buffer (50 mmol L−1 phosphate buffer, pH 7.4). The

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homogenate was then centrifuged at 12,000 × g for 10 min at 4 ◦ C, and the supernatant was collected. The reaction mixture contained 50 ␮L supernatant, 1 mL methylthymol blue solution (0.25 mol L−1 methylthymol blue, 0.6% polyvinyl pyrrolidone (PVP), 0.1 mol L−1 HCl), and 2 mL alkaline solution (0.2 mol L−1 Na2 SO3 , 0.1% (w/v) glycine, 0.23 mol L−1 NaOH). The absorbance of the solution was measured at 610 nm and the amount of Ca2+ was calculated from a standard curve prepared using dilutions of a 2.5 mmol L−1 calcium stock solution. Measurement of nitric oxide (NO) content The NO content in leaves was measured as described by Murphy and Noack (1994). Leaf tissue (0.50 g FW) was incubated with 100 U catalase and 100 U superoxide dismutase for 5 min to remove endogenous ROS, then 10 mL oxygenated hemoglobin (5 mmol L−1 ) was added and the mixture was incubated for 2 min before measuring absorbance at 550 nm. The amount of NO was determined based on the conversion of oxyhemoglobin into methemoglobin. Determination of phospholipase D (PLD) activity A standard curve for choline chloride was prepared according to the methods of Imamura and Horiuti (1978) and Shigeyuki and Yoshifumi (1978), with slight modifications, as follows: 100, 200, 300, 400, or 500 ␮L of a 1 mol L−1 choline chloride stock solution was added to 1.5 mL 10 mmol L−1 Tris–HCl buffer (pH 7.8), respectively, and then distilled water was replenished to the complete reaction mixture of the volume to 2 mL. The mixtures were incubated at 37 ◦ C for 20 min. PLD activity was measured in a 0.5-mL reaction mixture containing 100 ␮L 0.2 mol L−1 acetate (pH 5.5), 50 ␮L 0.1 mol L−1 CaCl2 , 100 ␮L 10 mmol L−1 lecithin emulsion, 165 ␮L distilled water, 50 ␮L enzyme solution, and 35 ␮L ether. The enzyme solution was added to initiate the reaction, and the mixture was incubated at 25 ◦ C for 20 min before adding 0.2 mL 1 mol L−1 Tris–HCl buffer solution containing 50 mmol L−1 EDTA, and 1.5 mL reaction buffer (Tris–HCl containing 3 U choline oxidase, 2 U peroxide enzymes, 15 ␮mol L−1 4-aminoantipyrine, and 21 ␮mol L−1 carbolic acid). The mixture was incubated at 37 ◦ C for 20 min and before reading absorbance at 500 nm. Determination of nitric oxide synthase (NOS) activity Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine, molecular oxygen, and nicotinamide adenine dinucleotide phosphate (NADPH) to NO, citrulline, and NADP+ . Measurement of NOS activity was carried out according to the method of Salter and Knowles (1998). NOS activity was quantified based on the conversion of oxyhemoglobin to methemoglobin by NO, which was monitored by measuring the change in absorbance at 401 nm. Determination of nitrate reductase (NR) activity Nitrate reductase (NR) activity was determined by measuring the formation of NO2 − . The extraction of the leaf material and the measurements of NR activity were conducted as described by Riens and Heldt (1992) with the following modifications. At indicated times, leaves were frozen in liquid nitrogen and then homogenized to a fine powder. The powder was stored in liquid nitrogen until analysis. For the activity assay, 1 mL medium containing 50 mmol L−1 Hepes-KOH (pH 7.5), 2 mmol L−1 dithiothreitol (DTT), 0.2% (v/v) Triton X-100, and MgC12 at indicated concentrations was added to the frozen leaf material (containing approximately 0.2 mg Chl). The solution was homogenized, and a

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100 ␮L-aliquot was added to 450 ␮L of the reaction medium, containing 40 mmol L−1 Hepes-KOH, 5 mmol L−1 KNO3 , 0.2 mmol L−1 NADH, and MgCl2 . The time between the addition of the medium to the frozen leaf material and the start of the assay was 2 min. For the enzyme assay, the mixture was incubated at 30 ◦ C for 2 min, and the reaction was terminated by adding 100 ␮L 1 mol L−1 zinc acetate solution. Then, 50 ␮L 0.15 mmol L−1 phenazine methosulfate was added to oxidize the un-reacted NADH. Activity was expressed as nmol NO2 − accumulated mg−1 cholophyll min−1 and per plant part. Determination of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity Frozen leaf tissue (0.5 g) was homogenized in 10 mL 50 mmol L−1 potassium phosphate buffer (pH 7.0) containing 1 mmol L−1 EDTA, 0.01% Triton X-100, and 1% PVP. The mixture was centrifuged at 15,000 × g for 20 min at 4 ◦ C. The NADPH oxidase activity in 0.1 mL supernatant was determined following the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H tetrazolium hydroxide by O2 − as described by Sagi and Fluhr (2001). Determination of phosphoenolpyruvate carboxylase (PEPC) activity Phosphoenolpyruvate carboxylase (PEPC) activity was measured as described by Giglioli-Guivarc’h et al. (1996). Leaf tissue (0.15 g) was homogenized in 1.5 mL extraction buffer [50 mmol L−1 Tris–HCl (pH 7.8), 5 mmol L−1 DTT, 1 mM of MgCl2 , 2% (w/v) PVP] using a mortar and pestle on ice. The homogenate was then centrifuged at 12,000 × g for 10 min at 4 ◦ C, and the supernatant was used for the assay. The reaction mixture consisted of 700 ␮L distilled water, 50 mmol L−1 Hepes-KOH (pH 8.0), 10 mmol L−1 sodium bicarbonate, 5 mmol L−1 magnesium chloride, 0.2 mmol L−1 nicotinamide adenine dinucleotide, 1.5 U malate dehydrogenase, 150 ␮L crude enzyme solution, 5 mmol L−1 phosphoenolpyruvate. All of the above reagents were added sequentially, and then the mixture was shaken before reading absorbance at 340 nm. The enzyme activity was calculated from the absorbance value.

Table 1 Genes and primers for RT-PCR. Gene

Primer

pepc

5 -AGCTCCACAGTTCGTCTGGT-3 (forward) 5 -GCTCAAGTGGCTCAAGGAAC-3 (reverse) 5 -CTGGGCCACACTACAATCCT-3 (forward) 5 -CAGCCTTGAAGTCCGATGAT-3 (reverse) 5 -CGCTGTACGGAGAGAAGGAG-3 (forward) 5 -ACTCGTGCATGATCCAGTTG-3 (reverse) 5 -CCCTCTTTCATCGGTATGGA-3 (forward) 5 -TTGATCTTCATGCTGCTTGG-3 (reverse)

Cu/Zn-SOD NAC6 Actin

ethanol dehydration, the paper was allowed to dry naturally before placing it in a liquid scintillation vial containing 10 ml distilled water for Cerenkov counting with an LS6500 scintillation counter (Beckman Coulter Inc., Fullerton, CA, USA). Protein kinase activity (cpm ␮g protein−1 min−1 ) was calculated as follows: protein kinase activity = value obtained from sample containing enzyme − value of blank control − background value (distilled water). Extraction of total RNA and quantitative PCR Total RNA was extracted using an RNA simple Total RNA Kit (Tiangen, Beijing, China), according to the manufacturer’s instructions. Reverse transcription reactions were conducted using a TaKaRa PrimeScript RT Master Mix Perfect Real Time Kit (TaKaRa, Dalian, China) and qRT-PCR analyses were conducted using an SYBR Premix Ex TaqTM II kit (TaKaRa), according to the manufacturer’s instructions. We used Applied Bio-systems instruments (Applied Biosystems, Foster City, CA, USA) for these analyses. The sequences of gene primers are shown in Table 1. Statistical analysis The data were analyzed by one-way ANOVA and LSD multiple comparison tests (p < 0.05) using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). qRT-PCR data were analyzed using the 2−Ct method. Results

Protein extraction and protein kinase activity assay in leaves Protein was extracted from leaves (0.1 g) in extraction buffer (2 mL) [100 mmol L−1 Hepes, pH 7.5, 5 mmol L−1 EDTA, 5 mmol L−1 ethylene glycol tetraacetic acid (EGTA), 15 mmol L−1 DTT, 15 mmol L−1 Na3 VO4 , 15 mmol L−1 NaF, 50 mmol L−1 ␤glycerophosphate (Sigma, St. Louis, MO, USA), 1 mmol L−1 phenylmethanesulfonyl fluoride, 5 mg mL−1 antipain (Sigma), 5 mg mL−1 aprotinin (Sigma), 5 mg mL−1 leupeptin (Sigma), 15% (w/v) glycerol] using the method of Zhang and Liu (2001) with minor modifications. After centrifugation at 18,000 × g for 15 min at 4 ◦ C, the supernatant was collected and then concentrated by centrifugation at 14,000 × g for 20 min in a Millipore (8–10 KD) concentrator. The supernatant was transferred to a clean tube, immediately frozen in liquid N2 , and stored at −75 ◦ C until analysis. The protein kinase activity assay was conducted as described by Zhang and Klessig (1997). The reaction mixture (40 ␮L) consisted of 25 mmol L−1 Tris, pH 7.5, 5 mmol L−1 MgCl2 , 1 mmol L−1 EGTA, 1 mmol L−1 DTT, 0.5 mg mL−1 histone-III, and 20 ␮L soluble protein solution. The mixture was incubated at 30 ◦ C for 10 min, then 2 ␮L 50 ␮mol L−1 ATP (contained 0.5 ␮Ci ␥-32 P-ATP) was added to start the reaction. After 10 min at 30 ◦ C, 42 ␮L 150 mmol L−1 H3 PO4 was added to terminate the reaction. A 20-␮L aliquot of the mixture was blotted onto a WHATMAN 3 filter paper strip (1 cm × 2 cm), and then the strip washed six times, each for 5 min, with 150 mmol L−1 H3 PO4 solution to completely remove the free ␥-32 P-ATP. After

PC showed higher RWC and PEPC activity under PEG-6000 treatment The changes in the RWC of WT and PC during the PEG-6000 treatment are shown in Fig. 1a. The RWC showed a smaller decrease in PC than in WT on day 1 of the PEG-6000 treatment. On day 2, the RWC of WT and PC decreased further (Fig. 1a). Throughout the PEG-6000 treatment, the RWC decreased less in PC than in WT. During the 2-day re-watering treatment, the RWC in PC recovered to the control level after 1 day, while that in WT took 2 days to recover to the control level (Fig. 1a). This result indicated that PC recovered faster than WT after the PEG-6000 treatment. In some stress conditions, CO2 promotes the activity of PEPC, which has a higher affinity for C than that of ribulose-1,5bisphosphate-carboxylase/oxygenase (Rubisco) (Pollastrini et al., 2014). We observed that the PEPC activity was much higher in PC than in WT during the PEG-6000 and re-watering treatments. The changes in RWC were similar to the changes in PEPC activities (Fig. 1b). Less inhibition of Pn in PC than in WT under PEG-6000 treatment When plants were treated with 15% PEG-6000, the Pn in WT decreased significantly. In contrast, in PC, there was no marked decrease in Pn on day 1, and only a small decrease in Pn on day 2.

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Fig. 1. RWC and PEPC activity of WT and PC under 15% PEG6000 treatment and rewatering for different days. (a) Changes of RWC both in WT and PC under 15% PEG6000 treatment (2 days) and rewatering (another 2 days), dates of each day were tested. (b) Changes of PEPC activity both in WT and PC under the same conditions as (a). Averages for three independent experiments are shown. Error bars represent standard deviations. Values indicated by the same letter do not differ significantly at the 5% level, as determined by ANOVA with Tukey’s test. “0” in X axis of this figure indicates the starting day just before the treatments. “1” and “2” in X axis of this figure indicates the first and second day of PEG treatment, respectively. “1 ” and “2 ” in X axis of this figure indicates the first and second day of rewatering after the PEG treatments, respectively. “WT-CK” indicates the WT plants without PEG treatment; “WT- PEG” indicates the WT plants under PEG treatments and then rewatering treaments. “PC-CK” indicates the PC plants without PEG treatment; “PCPEG” indicates the PC plants under PEG treatments and then rewatering treaments. Each treatment was replicated three times. Bars = SE. Bars with different letters were significantly different at the p < 0.05 (lowercase).

On day 1 of the re-watering treatment, the Pn partially recovered in both WT and PC. However, the Pn only recovered to its original level in PC (on day 2 of re-watering), whereas it did not recover to its original level in WT (Fig. 2a). Regulating stomata closure is the most direct and rapid response of suppression under drought stress. Both WT and PC were able to close stomata after day 1 of the PEG treatment, resulting in lower Gs. However, as the duration of the drought extended, the Gs value remained high in PC, and the stomata did not fully close (Fig. 2b). Compared with WT, PC still showed lower Ci values under the PEG6000 treatment (Fig. 2c). This finding suggested that the effects of PEG-6000 treatment on PC were a result of non-stomata limitation. Endogenous H2 O2 , Ca2+ , and NO participated in drought stress signaling in PC The changes in H2 O2 content differed between WT and PC under PEG-6000 stress (Fig. 3a). The H2 O2 content in PC was largely unchanged during the PEG-6000 and re-watering treatments. In contrast, the H2 O2 content increased markedly in WT, as a result

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Fig. 2. Pn, Gs and Ci of WT and PC under 15% PEG6000 treatment and rewatering for different days. (a) Changes of Pn both in WT and PC under 15% PEG6000 treatment (2 days) and rewatering (another 2 days), dates of each day were tested. (b) Changes of Gs both in WT and PC under the same conditions as (a). (c) Changes of Ci both in WT and PC under the same conditions as (a). Each treatment was replicated three times. Bars = SE. Bars with different letters were significantly different at the p < 0.05 (lowercase). For experimental details see Fig. 1.

of increased NADPH oxidase activity (Fig. 3b). The activity of APX (Fig. 3c) was elevated in PC during the drought and re-watering treatments (Fig. 3a). Also, the PLD activity was higher in PC than in WT during these treatments (Fig. 3d). Hydrogen peroxide is known to regulate endogenous Ca2+ levels. The production of H2 O2 during the oxidative burst requires Ca2+ influx, which activates the plasma membrane-localized NADPH oxidase. We measured the changes in Ca2+ concentrations in vivo, and observed differences between WT and PC (Fig. 4a). The Ca2+ concentration in WT increased on day 1 of the PEG-6000 treatment, but returned to the same level as that in the un-treated control on day 2. In PC, the Ca2+ concentration reached its highest level on day 2 of the PEG-6000 treatment. The Ca2+ concentration in PC returned to the same level as that in the untreated control in the next few days, during re-watering treatment.

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Fig. 3. H2 O2 contents, NADPH oxidase activity, APX activity and PLD activity of WT and PC under 15% PEG 6000 treatment and rewatering for different days. (a) H2 O2 content both in WT and PC for each day under 15% PEG6000 treatment (2 days) and rewatering (another 2 days). (b) NADPH oxidase activity both in WT and PC under the same conditions as (a). (c) APX activity both in WT and PC under the same conditions as (a). (d) PLD activity of WT and PC under the same conditions as (a). Each treatment was replicated three times. Bars = SE. Bars with different letters were significantly different at the p < 0.05 (lowercase). For experimental details see Fig. 1.

Fig. 4. Calcium ions (Ca2+ ) content, NO content, NOS activity and NR activity of WT and PC under 15% PEG 6000 treatment and rewatering for different days. (a) Calcium ions (Ca2+ ) contents both in WT and PC for each day under 15% PEG6000 treatment (2 days) and rewatering (another 2 days). (b) NO contents both in WT and PC under the same conditions as (a). (c) NOS activity both in WT and PC under the same conditions as (a). (d) NR activity of WT and PC under the same conditions as (a). Each treatment was replicated three times. Bars = SE. Bars with different letters were significantly different at the p < 0.05 (lowercase). For experimental details see Fig. 1.

B. Qian et al. / Journal of Plant Physiology 175 (2015) 9–20

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In plants, NO has been reported to stimulate increases in intracellular Ca2+ . The NO content in WT and PC increased under drought stress, compared with the levels in their respective untreated controls (Fig. 4b). In PC, the peak in NO content was later than the peak in Ca2+ content, suggesting that NO might be downstream of Ca2+ in the drought signaling pathway. The NOS activities in WT and PC were increased under PEG-6000 treatment, resulting in increased NO levels (Fig. 4c). There was an increase in NR activity in PC, but not WT, under the PEG-6000 treatment (Fig. 4d). This result suggested that the higher level of NO in PC than in WT could be related to both NOS and NR activities. Intermediate responses to drought stress: increased protein kinase activity and increased transcript levels of some transcription factor genes As mentioned above, ROS-mediated signaling involves protein phosphorrylation regulated by Ca2+ -dependent protein kinases. This implies that there is some crosstalk between Ca2+ and phosphorylation in regulating ROS production (Suzuki et al., 2011). Protein kinase activity increased in both WT and PC plants during the PEG-6000 and re-watering treatments (Fig. 5a). NAC6 is a drought-related transcription factor (Nakashima et al., 2007). Our data showed that during the drought treatment, the NAC6 transcript levels showed only minor changes in WT, but increased in PC. On day 2 of the PEG-6000 treatment, the transcript level of NAC6 in PC was 17.7 times that in its untreated control (Fig. 5b). The transcript level of C4 -pepc also increased in PC under PEG6000 treatment. On day 2 of the PEG-6000 treatment, the transcript level of C4 -pepc in PC was 3.7 times that in its untreated control (Fig. 5c). On days 1 and 2 of the re-watering treatment, the transcript levels of C4 -pepc remained high, at nearly 2.4 and 2.9 times that in the untreated control, respectively (Fig. 5c). The transcript levels of cu/zn-sod were significantly increased under the PEG-6000 treatment in both WT and PC. On day 1 of the PEG-600 treatment, the transcript levels in WT and PC were 1.6 and 3.7 times that in their respective untreated controls (Fig. 5d). The transcript levels of cu/zn-sod were slightly higher in PC than in WT. These results suggested that protein kinases and NAC6 increase the transcriptions of the maize C4 -pepc gene and some genes related to antioxidant defense. PC showed enhanced oxidative resistance as a result of higher antioxidant enzyme activities during drought Malondialdehyde (MDA) and the superoxide anion radical are important indices of lipid peroxidation in plants. Under PEG-6000 treatment, the MDA content (Fig. 6a) and superoxide anion radical concentration (Fig. 6b) significantly increased in both WT and PC. The increases were much greater in WT than in PC, indicating that WT suffered from greater oxidative stress than did PC under the PEG-6000 treatment. The activities of SOD (Fig. 6c), POD (Fig. 6d), and CAT (Fig. 6e) increased under the PEG-6000 treatment in WT and PC. The activities of all of these enzymes were higher in PC than in WT. This trend was also observed during the re-watering treatment (Fig. 6). Correlation analysis among parameters for PC and WT As shown in Tables 2 and 3, there were significant correlations between some pairs of indicators under PEG-6000 stress and rehydration. For WT, there was a significant relationship between Gs and SOD/POD (Table 2), implying that changes in Pn were due to stomata limitation. For PC, there were highly significant correlations between the following pairs of parameters: RWC and PEPC

Fig. 5. Changes of protein kinases activity, the genes expression (pepc, sod and NAC6) of WT and PC under 15% PEG6000 treatment and rewatering for different days. (a) Protein kinases activity both in WT and PC under 15% PEG6000 treatment (2 days) and rewatering (another 2 days), dates of each day were tested. (b) NAC6 relative expression both in WT and PC under the same conditions as (a). (c) pepc relative expression both in WT and PC under the same conditions as (a). (d) SOD relative expression both in WT and PC under the same conditions as (a). Each treatment was replicated three times. Bars = SE. Bars with different letters were significantly different at the p < 0.05 (lowercase). For experimental details see Fig. 1.

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Fig. 6. MDA content, superoxide anion radical production rate, antioxidant enzymes activity of WT and PC under 15% PEG6000 treatment and rewatering for different days. (a) MDA content both in WT and PC under 15% PEG6000 treatment (2 days) and rewatering (another 2 days), dates of each day were tested; (b) Superoxide anion radical production rate, both in WT and PC under the same conditions as (a). (c) SOD activity both in WT and PC under the same conditions as (a). (d) POD activity both in WT and PC under the same conditions as (a). (e) CAT activity both in WT and PC under the same conditions as (a). Each treatment was replicated three times. Bars = SE. Bars with different letters were significantly different at the p < 0.05 (lowercase). For experimental details see Fig. 1.

activity; Pn and APX activity; Pn and Ca2+ content; H2 O2 content and SOD activity; APX activity and Ca2+ content; and Ca2+ and APX (Table 3). These findings indicated that in PC, changes in Pn were more related to non-stomata limitation.

Discussion C4 -phosphoenolpyruvate carboxylase has been cloned and characterized in many plants, including rice (Ku et al., 1999), wheat (Hu et al., 2012), and Arabidopsis (Wang et al., 2012). Transgenic rice expressing maize pepc showed enhanced antioxidant capacity under drought stress (Fang et al., 2008). In this study, we observed that there was a smaller inhibition of photosynthesis in PC than in

WT under PEG-6000 treatment, and that PC showed better recovery than WT during re-watering treatment. In general, the RWC of plant tissues decreases under drought stress (Nayyar and Gupta, 2006). In this study, we observed that the decrease in RWC was smaller in PC than in WT on day 1 of the 15% PEG-6000 treatment. This may be related to the low Gs in PC. The RWC had recovered to its pre-drought level in both WT and PC by day 2 of the re-watering treatment. We also observed that the rate of decline in Pn was greater in WT than in PC under the PEG-6000 treatment. In PC, the Gs value remained high even on day 2 of the PEG-6000 treatment. The Pn showed better recovery in PC than in WT during the re-watering treatment. Therefore, it follows that WT was more seriously damaged than PC by drought. Compared with WT, PC may be able to achieve a better balance

Table 2 Correlation analysis of parameters in WT under 15% PEG6000 stress and rewatering for different days. Pn

Gs

SOD activity

POD activity

CAT activity

APX activity

H2 O2 content

Endogenous calcium

PEPC activity

Protein kinase activity

sod

NAC6

NO content

−0.55

−0.119 0.137

−0.191 −0.01 0.785

−0.124 0.039 −0.742 −0.934*

0.018 −0.101 −0.63 −0.923* 0.968**

0.119 0.285 0.687 0.790 −0.921* −0.926*

−0.718 0.202 0.243 0.674 −0.441 −0.588 0.316

−0.596 0.558 0.836 0.711 −0.56 −0.575 0.599 0.552

−0.502 −0.405 0.259 0.368 −0.058 −0.018 −0.275 0.539 0.27

−0.464 −0.456 0.011 0.087 0.22 0.256 −0.54 0.379 0.042 0.957*

0.114 0.425 −0.746 −0.638 0.477 0.322 −0.187 −0.268 −0.487 −0.762 −0.608

−0.648 0.459 −0.613 −0.592 0.775 0.612 −0.652 0.15 −0.132 0.055 0.237 0.522

0.282 −0.302 0.266 0.731 −0.835 −0.86 0.704 0.456 0.097 0.006 −0.19 −0.195 −0.653

0.671 0.351 0.752 0.354 0.561 0.457 0.547 0.699 −0.351 −0.897 0.354 0.698 0.498 0.397

Note: Correlation analysis of parameters in WT under 15% PEG6000 treatment (2 days) and rewatering (another 2 days). * P < 0.05. ** P < 0.01.

Table 3 Correlation analysis of parameters in PC under 15% PEG6000 stress and rewatering for different days.

RWC MDA content Pn Gs SOD activity POD activity CAT activity APX activity H2 O2 content Endogenous calcium PEPC activity pepc Protein kinase activity sod NAC6

MDA content

Pn

Gs

SOD activity

POD activity

−0.075

−0.73 −0.502

0.034 −0.482 0.566

0.345 0.757 −0.609 −0.050

0.421 0.749 −0.680 −0.094 0.995**

CAT activity 0.085 0.036 0.152 0.724 0.561 0.507

APX activity

H2 O2 content

Endogenous calcium

PEPC activity

pepc

Protein kinase activity

sod

NAC6

NO content

0.772 0.564 −.957* −0.353 0.754 0.814 0.059

0.469 0.783 −0.824 −0.327 0.945* .967** 0.292 0.896*

0.673 0.662 −0.945* −0.392 0.826 0.876 0.108 0.987** 0.953*

0.977** −0.239 −0.574 0.231 0.265 0.332 0.204 0.64 0.338 0.532

0.74 −0.281 −0.658 −0.333 0.011 0.089 −0.218 0.507 0.241 0.439 0.698

0.597 −0.547 −0.368 −0.278 −0.447 −0.364 −0.542 0.194 −0.2 0.076 0.581 0.853

0.178 0.703 −0.683 −0.826 0.286 0.339 −0.623 0.605 0.52 0.614 −0.03 0.136 0.123

0.861 0.957* −0.172 −0.514 −0.706 0.979* −0.315 −0.954* −0.026 0.214 0.070 0.537 −0.422 0.674 0.621 0.654 0.223 0.254 0.504 −0.951* 0.792 0.989* 0.832 0.955* 0.867 0.954* 0.406 0.879

B. Qian et al. / Journal of Plant Physiology 175 (2015) 9–20

RWC MDA content Pn Gs SOD activity POD activity CAT activity APX activity H2 O2 content Endogenous Calcium PEPC activity Protein Kinase activity sod NAC6

MDA content

Note: The Correlation analysis of parameters in WT under 15% PEG6000 treatment (2 days) and rewatering (another 2 days). * P < 0.05. ** P < 0.01.

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between water loss and photosynthesis. That is, in PC, the stomata aperture may be adjusted to minimize water loss via transpiration, while still allowing sufficient CO2 uptake to support photosynthesis. Clarifying the nature of the responses to drought might reveal important clues about the physiological basis of drought tolerance in PC. Many previous studies have shown that H2 O2 is involved in signaling during the response to diverse biotic and abiotic stresses (Gill and Tuteja, 2010). In Zea mays, H2 O2 accumulated in the subsidiary cells during drought stress and regulated stomata closure (Yao et al., 2013). However, H2 O2 can be a “double-edged sword”; that is, excessive H2 O2 can cause oxidative stress, which damages plant tissues. In this study, the total endogenous H2 O2 content did not increase in PC under drought stress. This was related to the high activities of enzymes that scavenge H2 O2 , such as APX, and lower activity of NADPH oxidase. In PC, stomata movement is regulated via phosphatidic acid (PA) and endogenous Ca2+ (Li et al., 2011). A previous study showed that this regulation of stomata movement in PC relied on low concentrations of H2 O2 , and PA could promote PLD activity independently of H2 O2 , and thus, increase PEPC activity and Pn (Ren et al., 2014). Based on these results, we speculated that the drought signal is not transmitted via H2 O2 in PC, unlike in WT. In PC, when the adverse environmental conditions ceased (i.e., re-watering after drought), H2 O2 was rapidly removed via its higher CAT and APX activities, thereby avoiding damage caused by excessive H2 O2 . Calcium plays an important physiological role as a second messenger. It regulates many physiological processes in plant growth and development, and is involved in responses to diverse environmental stresses (Dodd et al., 2010). Osmotic stress was shown to increase the intracellular Ca2+ concentration in guard cells (Siegel et al., 2009). However, elevated Ca2+ levels are potentially toxic to ATP metabolism, and Ca2+ can precipitate negatively charged biological molecules. Therefore, there is tight control of the cytosolic concentration ([Ca2+ ]) in plant cells (Bender and Sendden, 2013). We observed that [Ca2+ ] in WT and PC showed a transient peak under the drought treatment, but otherwise remained stable. However, the timing of the peak in [Ca2+ ] was earlier in WT than in PC. Interestingly, in PC, the peak in [Ca2+ ] coincided with the maximum transcript level of NAC6 and the peak in protein kinase activity. In plants, the release of NO by an NO donor induces a transient increase in [Ca2+ ]cyt in guard cells (Lamotte et al., 2006). This plays an important role in the drought resistance of maize (Zhang et al., 2011). Several studies on cell suspensions and/or tissues of various species have shown that NO donors can also trigger protein kinase activities as the downstream targets of the signaling molecules described above (Courtois et al., 2008). The production of NO mainly depends on NOS and NR pathways (Gupta et al., 2011). Distefáno et al. (2008) reported that NO can induce phosphatidic acid (PA) accumulation. In this paper, we observed that NOS activity was increased in both WT and PC under drought stress. Furthermore, the activity of NR and PLD activity in PC increased during the PEG and re-watering treatments, but not in WT. It was reported that the soluble sugars content is higher in PC than in WT (Li and Wang, 2013). In PC, the higher sugar levels combined with stress conditions can lead to increased NR activity, resulting in greater NO production. Hence, NO can induce PA via PLD in PC plant under PEG treatment. Protein kinases are an important class of regulatory factors in plant defense reactions (Wei et al., 2014). In particular, the membrane receptor protein kinases perceive the external environment stress signal, and their activity results in changes in the intracellular concentrations of several ions and molecules (Kulik et al., 2011). Thus, different protein phosphorylation pathways are activated, regulating the transcription and expression of downstream genes (Lee et al., 2011). Over-expression of the receptor protein kinase OsSIK1 in rice significantly enhanced CAT, SOD, and POD activities and

increased drought tolerance (Ouyang et al., 2010). In this study, we also observed that protein kinase activity was higher in PC than in WT. The increased protein kinase activity in PC coincided with the increase in NO activity after the transient increase in [Ca2+ ]. These findings indicated that there is an interaction among Ca2+ , NO, and protein kinases, especially under moderate drought conditions. Monreal et al. (2010) reported that PLD and PA were involved in the light-dependent up-regulation of phosphoenolpyruvate carboxylase-kinase (PEPCk) in leaves of sorghum. In this study, we also observed higher PLD activity in PC than in WT after the drought treatment. This finding suggested that there are some cross-talks among NO, PA, and protein kinases, which along with Ca2+ , regulate PEPC via phosphorylation. At present, little is known about the post-translational regulation of C4 -PEPC, for example, by phosphorylation. It is possible that phosphorylation of PEPC by Ca2+ -regulated kinases could represent a positive regulation mechanism. This topic deserves further study, as it could help to explain why PC plants are more tolerant than WT to PEG-6000 stress. Phosphoenolpyruvate carboxylase (PEPC) is present in all organisms capable of photosynthesis, including higher plants, algae, cyanobacteria, and photosynthetic bacteria. It is also present in most non-photosynthetic bacteria and protozoa, but not animals and yeast (Kai et al., 2003). In general, there are three types of PEPCs in plants, C3 -type, C4 -type, and root type (Mamedov et al., 2005). In rice, Osppc1, 2a, 2b, 3, and 4 encode PEPCs. Among these, Osppc 4 encodes a chloroplast PEPC that does not belong to any of the types described above (Masumotoa et al., 2010). Many studies have shown that compared with C3 plants, C4 plants have higher photosynthetic capacity and higher nitrogen- and water-use efficiencies under drought conditions. These advantages are closely related to PEPC in plants (Zhu et al., 2010). Transgenic plants expressing C4 pepc have been shown to have a high photosynthetic rate under photo-oxidation, high temperature, and drought conditions (Jiao et al., 2002, 2005; Bandyopadhyay et al., 2007; Ling et al., 2014). An increase in PEPC activity might be associated with increased gene transcription, protein synthesis, and/or protein phosphorylation (Doubnerva and Ryslava, 2011). The C4 -PEPC is regulated by phosphorylation by a PEPC-k. The light-dependent up-regulation of PEPC-k suggested that a Ca2+ -dependent protein kinase integrates the two second messengers, Ca2+ and PA (Monreal et al., 2010). Our RT-PCR data provided further evidence that the transcription of some genes such as C4 -pepc and cu/zn-sod are induced by PEG-6000 stress in PC. Therefore, we can speculate that in PC, the changes in concentrations of signaling molecules (down-regulation of H2 O2 , regulation of Ca2+ and NO) under drought stress may be related to the high level C4 -PEPC protein and C4 -PEPC transcripts. In signal transduction networks, including those leading to changes in gene expression after the perception of stress signals, different transcription factors and cis-transcriptional elements not only act as molecular switches for gene expression, but also the end of the process of signal transduction (Yamaguchi-Shinozaki and Shinozaki, 2006). NAC transcription factors are specific proteins with various functions in plant development and stress responses. In OsNAC6-overexpressing rice, 163 genes were up-regulated. These included genes encoding protein kinases, transcription factors, POD, and chitinase (Nakashima et al., 2007). Our RT-PCR data showed that in PC, NAC6 transcript levels were significantly enhanced, to approximately 17 times that in the untreated control, on day 2 of the PEG-6000 treatment. The higher expression of this transcription factor in PC may be related to the increased transcript levels of C4 -pepc and cu/zn-sod. Plants under drought stress can also be subjected to oxidative stress as a result of excess ROS generation. To overcome ROSinduced damage, plants have evolved well-developed and complex antioxidant mechanisms, including enzymatic and non-enzymatic defense systems. In this study, we observed that drought-stressed

B. Qian et al. / Journal of Plant Physiology 175 (2015) 9–20

plants were also subjected to oxidative stress, as reflected by increased levels of lipid peroxidation products (MDA and superoxide anion radicals). Furthermore, the activities of antioxidant enzymes such as SOD, POD, and CAT increased in both WT and PC under the PEG-6000 treatment. However, the extent of lipid peroxidation damage differed significantly between WT and PC. PC showed higher tolerance to oxidative stress, consistent with the results reported by Fang et al. (2008). We observed that APX activity was higher in PC on day 2 of the PEG-6000 treatment than in its untreated control. However, the APX activity in WT was not significantly different between the drought-treated and control plants. Since peroxidases eliminate H2 O2 , the increased activity of APX in PC may explain the lower H2 O2 levels. This enhanced oxidative tolerance in PC might because it has a greater ability to activate protein kinases and transcription factors via NO and Ca2+ . The cross-talk among protein kinases, NO, and Ca2+ in PC under drought stress is worthy of further study. The deployment of multiple positive and negative regulatory mechanisms at different stages of the drought treatment demonstrates the critical importance of balancing these responses. Overall, the improved drought tolerance of PC is associated with elevated PEPC activity and changes in the levels of various signaling molecules under drought stress; in PC, up-regulation of NO and Ca2+ regulation induces higher protein kinase activity for up-regulation of PEPC activity, enhances upstream gene expression of the transcription factor, such as NAC6, then induces higher downstream gene expression C4 -pepc, and cu/zn-sod, which encode components of the antioxidant defense system. These responses in PC enhance the antioxidant system that scavenges ROS at a cellular level, reducing the inhibition of Pn and allowing leaves to retain higher RWC under drought conditions.

Conclusion The molecular mechanism of drought tolerance in PC is related to the signaling processes via NO and Ca2+ involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4 pepc, and some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 30871459 and No. 31371554), the National Scientific and Technological Support Projects (2009BAC63B01), the Agricultural Science and Technology Innovation Fund of Jiangsu in China [CX(14)5004], Jiangsu Key Laboratory for Microbes and Functional Genomics, the Ministry of Environmental Protection National Commonweal Research Project (201009023), and the Natural Science Foundation of Jiangsu Province (No. BK20130708). The authors thank the anonymous reviewers and editorial staff for their time and attention.

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