Cold priming drives the sub-cellular antioxidant

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May 20, 2014 - the carbohydrate metabolism and photosynthetic properties. (Ruelland et al. ... stress, plants have developed some defense mechanisms to enhance their ... enging systems in chloroplasts and mitochondria to cold priming treatment and ..... than did the NL plants for both cultivars, indicating that the cold.
Plant Physiology and Biochemistry 82 (2014) 34e43

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Research article

Cold priming drives the sub-cellular antioxidant systems to protect photosynthetic electron transport against subsequent low temperature stress in winter wheat Xiangnan Li a, b, Jian Cai a, **, Fulai Liu b, Tingbo Dai a, Weixing Cao a, Dong Jiang a, * a National Engineering and Technology Center for Information Agriculture/Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China b University of Copenhagen, Faculty of Science, Department of Plant and Environmental Sciences, Højbakkegaard All e 13, DK-2630 Taastrup, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 January 2014 Accepted 12 May 2014 Available online 20 May 2014

Low temperature seriously depresses the growth of wheat through inhibition of photosynthesis, while earlier cold priming may enhance the tolerance of plants to subsequent low temperature stress. Here, winter wheat plants were firstly cold primed (5.2  C lower temperature than the ambient temperature, viz., 10.0  C) at the Zadoks growth stage 28 (i.e. re-greening stage, starting on 20th of March) for 7 d, and after 14 d of recovery the plants were subsequently subjected to a 5 d low temperature stress (8.4  C lower than the ambient temperature, viz., 14.1  C) at the Zadoks growth stage 31 (i.e. jointing stage, starting on 8th April). Compared to the non-primed plants, the cold-primed plants possessed more effective oxygen scavenging systems in chloroplasts and mitochondria as exemplified by the increased activities of SOD, APX and CAT, resulting in a better maintenance in homeostasis of ROS production. The trapped energy flux (TRO/CSO) and electron transport (ETO/CSO) in the photosynthetic apparatus were found functioning well in the cold-primed plants leading to higher photosynthetic rate during the subsequent low temperature stress. Collectively, the results indicate that cold priming activated the subcellular antioxidant systems, depressing the oxidative burst in photosynthetic apparatus, hereby enhanced the tolerance to subsequent low temperature stress in winter wheat plants. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Cold priming Electron transport Chloroplast Mitochondria Antioxidant defense Wheat (Triticum aestivum L.)

1. Introduction As one of the most critical abiotic stresses limiting growth and productivity of winter wheat (Triticum aestivum L.), low temperature seriously depresses plant growth and causes significant reduction in grain yield (Kosova et al., 2013). It induces a series of metabolic changes, including inactivation of many metabolic enzymes and disturbance of the metabolic regulations (Xu et al., 2012), accumulation of osmolytes (e.g. proline, glycinebetaine)

Abbreviations: APX, ascorbate peroxidase (EC 1.11.1.11); BSA, bovine serum albumin; CAT, catalase (EC 1.11.1.6); CS, cross-section; DTT, dithiothreitol; EGTA, ethylene glycol tetra acetic acid; FNR, ferredoxin-NADPþ reductase; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; MOPS, 3-(N-morpholino) propanesulfonic acid; PMSF, phenylmethylsulphonyl fluoride; PVP, polyvinylpyrrolidone; RC, reaction centre; ROS, reactive oxygen species; SLA, specific leaf area; SOD, superoxide dismutase (EC 1.15.1.1). * Corresponding author. Tel./fax: þ86 25 8439 6575. ** Corresponding author. Tel./fax: þ86 25 8439 5478. E-mail addresses: [email protected] (J. Cai), [email protected] (D. Jiang). http://dx.doi.org/10.1016/j.plaphy.2014.05.005 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.

(Thakur and Nayyar, 2013; Ruelland et al., 2009), modifications of the carbohydrate metabolism and photosynthetic properties (Ruelland et al., 2009; Crosatti et al., 2013). Photosynthesis converts light energy into ATP and redox equivalents (NADPH), which is the primary metabolic sink for plant growth (Ruelland et al., 2009). Low temperature stress affects many aspects of photosynthesis. For instance, it can inhibit thylakoid electron transport by increasing membrane viscosity and restricting the diffusion of plastoquinone. Light energy is trapped by the antenna of PSI and PSII to drive the charge separation in the reaction centres (RCs). This process can be disturbed by low temperatures, since the chlorophyll antenna complexes are able to trap more energy than the capacity of biochemical procession in the photosynthetic RCs, resulting in over-energized status in the thylakoid membranes (Ensminger et al., 2006). One of the consequences of this over-energized state is photodamage due to overproduction of reactive oxygen species (ROS) (Ruelland et al., 2009; Ashraf and Harris, 2013). It has been documented that the activities of the scavenging enzymes are depressed by low temperature stress (Thakur and Nayyar, 2013; Ruelland et al., 2009; Li et al.,

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2013), leading to inefficiency in counterbalancing the ROS production in the mitochondrial and chloroplastic electron transfer reactions (Ruelland et al., 2009). To survive under low temperature stress, plants have developed some defense mechanisms to enhance their tolerance to the stress (Thakur and Nayyar, 2013). Priming, defined as a temporally limited experience of an environmental stimulus which prepares the plant to cope more successful with a future environmental stimulus, has been find in wheat plants under varied abiotic stresses, including heat (Wang et al., 2014) and waterlogging (Li et al., 2011). However, the priming induced responses to cold stress remain largely elusive. Higher plants possess active oxygen scavenging systems including several antioxidant enzymes, such as superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6), and the non-enzymatic antioxidants (Thakur and Nayyar, 2013). It is well known that the oxygen scavenging systems activated by cold hardening play a key role in enhancing cold tolerance (Thakur and Nayyar, 2013), especially those in chloroplasts and mitochondria, the major sites of ROS production in plant cells (Crosatti et al., 2013; Jacoby et al., 2012). However, little is known about the responses of the oxygen scavenging systems in chloroplasts and mitochondria to cold priming treatment and their roles in improving low temperature tolerance in wheat. In the present study, wheat plants were firstly cold-primed for 7 d, and after two weeks recovery the plants were then exposed to a 5 d low temperature stress. The changes of oxygen scavenging systems in chloroplasts and mitochondria were investigated during the cold priming and the low temperature stress periods. It was hypothesized that modulations of the antioxidant systems in chloroplasts and mitochondria are involved in the acquirement of low temperature tolerance induced by the cold priming in winter wheat plants. The results will be helpful in understanding the roles of antioxidant systems in enhancing cold tolerance in winter crops.

2. Materials and methods 2.1. Plant materials and treatments A semi-field experiment was conducted in the open-top chamber (OTC, Southeast Co. Ltd, Ningbo, China) at Lianyungang Experimental Station (119 320 E, 34 300 N) of Nanjing Agricultural University, Jiangsu Province, China during the wheat growing season in 2010e2011. Two winter wheat cultivars were used, i.e. a cold susceptible cultivar Funo and a cold tolerant cultivar HM18 (Data of preliminary experiment not published). The seeds of these two cultivars were sowed in the chamber on 16th October 2010. Before sowing, 120 kg N ha1, 60 kg P2O5 ha1 and 120 kg K2O ha1 were applied as basal fertilizer and another 120 kg N ha1 was topdressed after jointing. The experimental design is shown in Fig. 1. In brief, a 7 d cold priming where 5.2  C lower than the ambient

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temperature (10.0  C) was conducted at the Zadoks growth stage 28 (Zadoks et al., 1974) (plants possessed one main shoot and eight tillers, starting on 20th of March), and after 14 d recovery a 5 d low temperature stress was imposed in which the temperature was 8.4  C lower than the ambient temperature (14.1  C) at the Zadoks growth stage 31 (Zadoks et al., 1974) (jointing stage, i.e. the 1st node was detectable, starting on 8th April). The detailed temperature data during the cold priming and subsequent low temperature stress was shown in Fig. 2. Low temperature treatments were applied with the OTC system. Air was cooled by a compressor (5.5 kW, CC-107, Ningbo Southeast Co., China), and then the precooled air was driven by an air blower (350 W, AH9, Ningbo Southeast Co., China) through a major ducting tube connected with the sub-ducting tubes. In the sub-ducting tubes, small holes were drilled with uneven distance to make sure that very similar volume of pre-cooled air was released by each hole (the hole distance in the sub-conducting tube was longer near the major conducting tube while was shorter at the distal end). Six temperature and humidity sensors were installed to record the real-time data at a 10-min interval in the treatment and reference chambers, respectively. Finally, three treatments were conducted; non-cold priming þ low temperature stress (NL), cold priming þ low temperature stress (CL), non-cold priming þ no low temperature stress (NN). The experiment was a split-plot design with temperature treatment as the main plot and wheat cultivar as subplot, and with three replicates for each treatment. Samples and measurements were conducted just before the onset and just after the ending of the cold priming and the low temperature stress, and after 5-day recovery of the low temperature stress. 2.2. Shoot biomass, concentrations of N and chlorophyll in the last fully expanded leaves Five wheat plants were harvested for each replicate. The harvested shoot samples were oven-dried to get shoot biomass. Fresh leaf (0.1 g) was sliced and incubated in 50 ml of pigment extraction solution containing acetone and anhydrous ethanol (1: 1, v/v) in dark at 25  C for 12 h. The supernatant was collected and the absorbance measured at 663 nm and 647 nm. Total chlorophyll concentration was then calculated according to Arnon (1949). Concentrations of total nitrogen in dry leaf samples were measured using the Kjeldahl method (Zhang et al., 2011). 2.3. Gas exchange, Chl a fluorescence transient and leaf area of the last fully expanded leaves Photosynthetic rate (PN) and stomatal conductance (gs) of the latest fully expanded leaf were measured with a portable photosynthesis system (LI-6400, LI-Cor, NE, USA) at a CO2 concentration of about 380 mmol mol1, at a photosynthetically active radiation of 1200 mmol m2 s1. On each measurement occasion, five leaves

Fig. 1. Schematic representation of experimental design and treatments. The morphology date was indicated following the Zadoks scale: Stage 28 indicates plants possessing one main shoot and eight tillers; Stage 31 indicates the 1st detectable node emerges (Jointing stage). CL refers to cold priming þ spring low temperature treatment without cold priming; NL refers to spring low temperature treatment; NN refers to the normal temperature control.

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Fig. 2. Daily mean temperatures in the CL and NN treatments during the cold priming and the later spring low temperature. CL refers to cold priming þ spring low temperature treatment; NL refers to spring low temperature treatment without cold priming; NN refers to the normal temperature control.

were taken for each treatment. Fast Chl a fluorescence induction curve (OJIP curve) was measured using the same leaf as for the gas exchange analysis with a Plant Efficiency Analyzer (Pocket-PEA, Hansatech, Norfolk, UK). Before measuring, leaves were darkadapted for 0.5 h. The collected data were processed by the program PEA Plus 1.04. 2.4. Chloroplasts and mitochondria isolation and enzyme activity analysis Mitochondria and chloroplasts in the last fully expanded leaves €diger et al. were isolated and purified following the protocol of Ro (2010). Briefly, leaf samples (6 g) were ground in 30 ml extraction buffer (0.45 M sucrose, 15 mM 3-(N-morpholino) propanesulfonic acid (MOPS), 1.5 mM ethylene glycol tetra acetic acid (EGTA), 0.6% polyvinylpyrro-lidone (PVP), 0.2% bovine serum albumine (BSA), 0.2 mM phenylmethylsulphonyl fluoride (PMSF) and 10 mM dithiothreitol (DTT)). Homogenate was filtered through eight layers of gauze and then the filtrate was centrifuged at 2000 g for 5 min. The supernatant was collected for mitochondria isolation, while the sedimentation was used for chloroplast isolation. For the mitochondria isolation, the supernatant was centrifuged at 6000 g for 5 min followed by 16 000 g for 10 min. Then, the sedimentation was carefully re-suspended in 1 ml washing buffer (0.3 M sucrose, 10 mM MOPS, 1 mM EGTA and 0.2 M PMSF) and layered on the top of a 6 ml layered system (18%, 23%, 40% Percoll) for the step gradients. After centrifugation at 12 000 g for 45 min, the crude mitochondria in the lower band was carefully

collected with a pipette, and washed twice with washing buffer followed by centrifugation at 12 000 g for 15 min. The intact chloroplasts were re-suspended in 500 ml of re-suspension buffer (0.4 M mannitol, 10 mM MOPS, 1 mM EGTA and 0.2 M PMSF) and kept at 4  C until use. For the chloroplast isolation, the sedimentation was re-suspended with sorbitol re-suspension medium (SRM, 0.33 M sorbitol in 50 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES)), and then layered on the top of a 7 ml layered system (35%, 80% Percoll) for the step gradients. The chloroplasts were collected and washed with 2 ml SRM followed by centrifugation at 1100 g for 10 min. Finally, the intact chloroplasts were kept with 2 ml SRM at 4  C. The concentrations of chlorophyll and soluble protein were determined using the method of Arnon (1949) and Zheng et al. (2009), respectively. Following the methods of Zheng et al. (2009), H2O2 concentration was measured by monitoring the absorbance of titanium peroxide complex at 410 nm. APX activity was determined by monitoring the decrease at 290 nm, and the activity of SOD was measured by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) (Yang et al., 2007). CAT activity was measured as described by Tan et al. (2008). Three replicates were measured for each treatment. 2.5. Statistical analysis All data were firstly tested for homogeneity of variance with boxplot and then subjected to two-way ANOVA to determine the

Table 1 Responses of shoot biomass, N concentration, chlorophyll concentration and leaf area of last fully expanded leaves to cold priming and spring low temperature stress in winter wheat. Cultivar

Cold priming

Spring low temperature stress

Treatment

Shoot biomass (g plant1)

Leaf N concentration (g m2)

Chlorophyll concentration (mg g1)

Leaf area (cm2 leaf1)

Treatment

Shoot biomass (g plant1)

Leaf N concentration (g m2)

Chlorophyll concentration (mg g1)

Leaf area (cm2 leaf1)

Funo

C

0.78 ± 0.01c

1.66 ± 0.06a

5.40 ± 0.23ab

17.74 ± 0.55a

HM18

N C

0.79 ± 0.01bc 0.81 ± 0.01ab

1.75 ± 0.04a 1.68 ± 0.07a

6.25 ± 0.17a 5.35 ± 0.24ab

17.26 ± 0.39a 13.55 ± 0.35b

N

0.82 ± 0.01a 84.78* 0.94 0.04

1.75 ± 0.04a 0.13 2.15 0.03

6.08 ± 0.13a 0.43 12.80* 0.07

13.99 ± 0.40b 48.94* 0.01 3.46

CL NL NN CL NL NN

0.96 ± 0.98 ± 1.04 ± 0.86 ± 0.85 ± 0.86 ± 32.34* 1.28 1.31

1.88 ± 0.02b 1.75 ± 0.04c 2.03 ± 0.06a 1.73 ± 0.02c 1.75 ± 0.06c 1.85 ± 0.04bc 99.18** 10.19** 2.46

4.76 ± 0.14b 4.90 ± 0.11b 6.60 ± 0.11a 4.88 ± 0.12b 4.87 ± 0.04b 6.38 ± 0.11a 0.09 236.47** 1.88

13.78 ± 13.08 ± 21.20 ± 15.01 ± 15.25 ± 16.85 ± 0.11 21.99** 9.11**

FC FT FCT

0.02ab 0.02ab 0.06a 0.03bc 0.01c 0.02c

0.23b 1.01b 1.52a 0.14b 0.49b 0.67ab

C and N refers to cold priming and non-cold priming, respectively. CL refers to cold priming þ spring low temperature treatment; NL refers to spring low temperature treatment without cold priming; NN refers to the normal temperature control. Data are means ± SE (n ¼ 3). Different small letters in the same column refer to significant difference between treatments at P < 0.05 level. FC, FT and FCT indicates F-value of cultivar, treatment and interaction of cultivar by treatment, respectively. * and ** refers to significant level of 0.05 and 0.01, respectively.

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Fig. 3. Responses of net photosynthetic rate (PN) and stomatal conductance (gs) of last fully expanded leaves to cold priming and spring low temperature stress in winter wheat. CL refers to cold priming þ spring low temperature treatment; NL refers to spring low temperature treatment without cold priming; NN refers to the normal temperature control. Data are means ± SE (n ¼ 3).

significant differences between treatments and cultivars using the software of SPSS (Ver. 10.0 SPSS, Chicago, IL, USA). The data collected before and after cold priming and the data collected before and after the subsequent low temperature stress were analyzed separately. Energy pipeline leaf model of phenomenological fluxes (per cross-section, CS) was performed using the Biolyzer 3.0 software (Bioenergetics Lab., Geneva, Switzerland).

3. Results 3.1. Plant growth and leaf N and chlorophyll concentrations The two-way ANOVA analysis shows that shoot biomass was not significantly affected by the cold priming (C), but differed significantly between the two cultivars under both cold priming and low temperature stress (Table 1). The N concentration and area of the last fully expanded leaf were also unaffected by cold priming, whereas the total chlorophyll concentration was decreased by cold priming. After the subsequent spring low temperature stress (L), leaf chlorophyll concentration and leaf area were lowered in the cold-primed (CL) and non-cold primed (NL) plants as compared to the control (NN) plants for both cultivars. In Funo, leaf N concentration was significantly higher in the CL than in the NL plants, while it was identical between the CL and NL plants for HM18. In addition, no differences in shoot biomass, chlorophyll concentration and leaf area were found between the CL and NL plants for both cultivars.

3.2. Photosynthetic rate (PN) and stomatal conductance (gs) In relation to the non-cold-primed plants, PN and gs of the last fully expanded leaf decreased with cold priming for both cultivars (Fig. 3). PN was significantly depressed by the subsequent low temperature stress as compared with NN. However, PN was much higher in CL than in NL and after the 5 d recovery. In should be noted that PN was fully recovered to the level of NN after five days of removal of the low temperature stress in CL plants in HM18, while it was only partially recovered in CL plants in Funo, and in NL plants in both cultivars. gs was depressed during cold priming and later spring low temperature stress. However, gs only partially recovered after the removal of the low temperature stress, and were similar between CL and NL plants. The recovery of gs seemed better in HM18 than in Funo, but no significant difference was found between the two cultivars. 3.3. Antioxidant system in chloroplasts and mitochondria In chloroplast, the H2O2 concentration was significantly higher in the cold-primed plants compared to the plants under normal temperature (Fig. 4, P < 0.01), and then slightly decreased during the 14-day recovery period for both cultivars. However, it was still higher in the cold-primed plants than the non-primed plants after the recovery. In accordance, activities of SOD, CAT and APX were significantly improved by the cold priming. However, the activities of these enzymes showed much quicker recovery than did H2O2 concentration. After 14-day recovery, activities of SOD and CAT

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Fig. 4. Responses of concentrations of H2O2, activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) in chloroplasts of last fully expanded leaves to cold priming and spring low temperature stress in winter wheat. CL refers to cold priming þ spring low temperature treatment; NL refers to spring low temperature treatment without cold priming; NN refers to the normal temperature control. Data are means ± SE (n ¼ 3).

were fully recovered in both varieties, activity of APX were fully recovered in Funo whilst it was only partially recovered in HM18. The 5 d low temperature stress caused larger increase of H2O2 concentration than the cold priming did, and the increment of H2O2 concentration was significantly less in CL than in NL plants. After

recovery, H2O2 concentration decreased in CL plants, while it was still maintained at very high concentration in NL plants. SOD activity was also significantly increased by low temperature stress and was maintained a high level after recovery. However, SOD activity was much higher in CL than in NL plants. APX activity was

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Fig. 5. Responses of concentrations of H2O2, activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) in mitochondria of last fully expanded leaves to cold priming and spring low temperature stress in winter wheat. CL refers to cold priming þ spring low temperature treatment; NL refers to spring low temperature treatment without cold priming; NN refers to the normal temperature control. Data are means ± SE (n ¼ 3).

also stimulated by low temperature stress, and was significantly higher in CL than in NL plants (P < 0.01). After recovery, APX activity decreased, and was higher in NL than in CL plants for Funo; and the reverse was the case for HM18. CAT activity was also significantly increased by the spring low temperature stress; however, unlike

SOD and APX, it was higher in NL than in CL plants. After recovery, CAT activity in both the NL and CL plants was fully restored to the level of NN plants. Mitochondrial H2O2 concentration was also significantly increased by both the cold priming and later spring low

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temperature stress (Fig. 5). H2O2 concentration was lower in CL plants and was recovered faster after removal of the low temperature stress as compared with the NL plants. Nonetheless, it remained significantly higher in CL and NL plants in relation to NN plants after recovery. Compared to the non-cold-primed plants, SOD activity was slightly increased in Funo while significantly increased in HM18 after cold priming; it was recovered to a similar level as of the control plants two weeks after removal of cold priming. In Funo, SOD activity was significantly increased by low temperature stress, and was higher in CL than in NL plants; after recovery, it was restored quickly in NL plants, whereas it was remained at higher level in CL as compared to NL and NN plants. In HM18, SOD activity was significantly increased only in CL plants and maintained at higher level than NL plants after recovery. In Funo, compared to the NN plants, APX activity was increased significantly to a similar level in both NL and CL plants. After recovery, the activity of APX was significantly lower in CL than in NL plants. In HM18, APX activity in the cold-primed plants was significantly higher than the non-cold-primed plants, while it was significantly higher in CL than in NL plants even after recovery. In Funo, CAT activity showed a similar pattern as for SOD activity. In HM18, CAT activity was increased in both NL and CL plants in relations to NN plants, and maintained at higher level after recovery; however, no significant difference in CAT activity was found between NL and CL plants even after recovery. 3.4. Chlorophyll fluorescence The maximum quantum yield of the PSII (FV/FM) were significantly lower in CL and NL plants than in NN plants; however, for both cultivars the reduction in FV/FM was more pronounced in NL than in CL (Fig. 6, P < 0.01). A similar pattern of change was found in the ratio of the efficiency of electron donation to PSII (FV/FO). The interpretations of the selected JIP-test parameters based on the fluorescence rise transients under different treatments are shown in Table 2. It was found that in both cultivars the maximum quantum yield for primary photochemistry (4PO), quantum yield for electron transport (ET) (4EO), probability that an electron moves further than QA (jEO) and quantum yield for reduction of end electron acceptors at the PSI acceptor side (RE) (4RO) in CL and NL plants were lower as compared to the NN plants. In addition, in both cultivars they were all significantly higher in CL than in NL plants. The derived parameters from the OJIP curves were summarized by means of energy pipeline leaf model of phenomenological fluxes

Table 2 Explanations of selected JIP-test parameters used in the present study. Fluorescence parameters FO FV ¼ FT  FO FM 4PO 4EO 4RO

jEO TRO/CSO ETO/CSO REO/CSO ABS/CSO DIO/CSO

Minimal fluorescence, when all PSII RCs (reaction centers) open Variable fluorescence at time t Maximal recorded fluorescence intensity Maximum quantum yield for primary photochemistry Quantum yield for electron transport (ET) Quantum yield for reduction of end electron acceptors at the PSI acceptor side (RE) Probability that an electron moves further than QA Trapped energy flux per CS Electron transport flux per CS PSI acceptor per CS Absorption flux per CS Non-photochemical quenching per CS

Subscript “O” indicates that the parameter refers to illumination onset, when all RCs are assumed to be open.

(per cross-section, CS) as shown in Fig. 7. In both cultivars, the trapped energy flux per CS (TRO/CSO) was increased in NL plants and decreased in CL plants as compared with that in NN plants. Electron transport flux per CS (ETO/CSO) and the number of active RCs in PS II cross-section (shown by open circles in Fig. 7) were higher in CL than in NL plants; however, in both cultivars they were lower in CL and NL plants as compared to those of the NN plants. PSI acceptor per CS (REO/CSO) was much lower (33.1%) in NL plants, while slightly higher (3.4%) in CL as compared to NN plants for Funo. For HM18, REO/CSO in NL and CL plants was 18.6% and 5.4% lower, respectively, than in NN plants. In addition, the energy absorbed per excited CS (ABS/CS) in NL and CL plants was significantly higher than in NN plants for both cultivars. 4. Discussion In the present study, wheat plants were firstly cold-primed and subsequently subjected to a 5 d low temperature stress after two weeks recovery at ambient temperature. The results showed that cold priming did not affect concentrations of leaf N and chlorophyll, and leaf area. However, the subsequent low temperature stress significantly affected these traits in the leaves, being that CL and NL treatments significantly lowered leaf area, concentrations of N and chlorophyll in the last fully expanded leaf as compared with the NN treatment. Consistent with this, decreased chlorophyll concentration has been found in wheat leaves exposed to low temperature stress (Liu et al., 2012). Valluru et al. (2012) reported that chilling

Fig. 6. Fluorescence transient chlorophyll a parameters deduced from analysis of the JIP-test of wheat leaves after spring low temperature. The chlorophyll a fluorescence curves for all treatments were measured at 12th April. Each parameter is expressed as fraction relative to the values of the control. CL refers to cold priming þ spring low temperature treatment; NL refers to spring low temperature treatment without cold priming; NN refers to the normal temperature control. Data are means ± SE (n ¼ 3).

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Fig. 7. Energy pipeline leaf model of phenomenological fluxes (per cross-section, CS) in last fully expanded leaves after spring low temperature in winter wheat. The interpretation of ABS/CSO, TRO/CSO, ETO/CSO and DIO/CSO are shown in Table 2. CL refers to cold priming þ spring low temperature treatment; NL refers to spring low temperature treatment without cold priming; NN refers to the normal temperature control. Data are means ± SE (n ¼ 3). Each relative value is drawn by the width of the corresponding arrow, standing for a parameter. Empty and full black circles indicate, respectively, the percentage of active (QA reducing) and non-active (non-QA reducing) reaction centers of PSII.

stress also decreased the size and biomass of flag leaf, and resulted in smaller specific leaf area (SLA) in wheat, which was in agreement with our findings. Moreover, the cold susceptible cultivar Funo had significantly larger reductions in shoot biomass, N concentration and leaf area of the last fully expanded leaves than did the cold tolerant cultivar HM18. Photosynthesis is a highly sensitive process to any change in environmental conditions, especially extreme temperatures, because it is essential to maintain the balance between light energy absorbed by photosystems and energy consumed by metabolic sinks in the plant (Ensminger et al., 2006). In the present study, PN was significantly depressed by low temperatures during both the cold priming and the subsequent low temperature stress treatment. Nonetheless, CL plants showed a better photosynthetic performance in relation to NL plants as exemplified by higher leaf PN and gs (Fig. 3). In addition, it was very interesting that the reduction in PN was more pronounced than the reduction in gs in NL plants in relation to CL plants. This indicated a better protection of leaf function in the CL plants than in NL plants, which could be attributed not only to the adjustment of stomatal openness but also to the regulation the non-stomatal processes. In the cold tolerant cultivar HM18, PN of CL plants could recover to the control level after recovery, while it was not the case in the cold susceptible cultivar Funo, indicating that the cold priming exerted greater influence on the tolerance to low temperature stress in terms of photosynthetic performance in the cold tolerant cultivar. It is well known that low temperature stress exacerbate the imbalance between the excessively absorption and the limited photo-chemical utilization capacity of the light energy, and adjustments of photosynthesis process are necessary to maintain the balance of energy flow in order to cope with low temperature stress (Ensminger et al., 2006). The fast chlorophyll a fluorescence induction curve has been widely used to investigate the photosynthetic electron transport processes under abiotic stresses (Strasser et al., 2004). Here, the CL plants possessed higher FV/FM and FV/FO than did the NL plants for both cultivars, indicating that the cold priming enhanced the quantum yield of the PSII and the electron  donation to PSII during the later low temperature stress (Spoustova et al., 2013). The efficiency balance of the dark reactions after QA expressed as jEo (Strasser et al., 2004), was found also higher in CL

than in NL plants. 4EO indicates the maximum quantum yield for electron transport beyond QA and also represents the maximum quantum yield of primary photochemistry; while 4PO reflects the efficiency of the light reactions (Chen et al., 2011). In this study, these parameters were maintained at a relatively higher level in CL plants as compared to the NL plants, indicating that the low temperature damage to the primary photochemistry reaction was minimized by cold priming. Between the two cultivars, values of these parameters in the CL and NL plants decreased to a lesser extent in the cold tolerant cultivar HM18 than the cold susceptible cultivar Funo. In addition, a higher relative value of 4RO in the CL compared to the NL plants suggested that cold priming could have induced protective effect on the end acceptors at the PSI electron acceptor side as well as the activity of ferredoxin-NADPþ reductase (FNR) in wheat plants under subsequent low temperature stress (Chen et al., 2012). The derived parameters from the chlorophyll a fluorescence induction curve can be further visualized by the dynamic energy pipeline leaf model of the photosynthetic apparatus (Strasser, 1987; Mehta et al., 2010). The leaf model of phenomenological energy fluxes (per cross-section) (Lu and Vonshak, 1999) was used in the present study as shown in Fig. 7. Electron transport in a PS II crosssection (ETO/CSO) presented the reoxidation of reduced QA via electron transport over a cross-section of active and inactive RCs (Mehta et al., 2010; Force et al., 2003). The density of the active RCs in PS II cross-section (indicated as open circles in Fig. 7) was higher in CL plants in relation to NL plants, implying that cold priming allowed less active state RCs were converted into inactive state RCs to reduce the overproduction of electron via the photo-chemical processes under subsequent low temperature stress. It is interesting that an increase in the energy absorption per excited crosssection (ABS/CSO) and a decrease in non-photochemical quenching (DIO/CSO) were observed in the CL and NL plants as compared with the NN plants, indicating that the energy absorption efficiency of PS II was not reduced under low temperature stress, and maintenance of the thermo-stability of PSII under abiotic stress was achieved by increasing non-photochemical quenching (Niyogi and Truong, 2013). The main sources of ROS production include the photosynthesis process in chloroplasts, the photorespiration in

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peroxisomes and the respiration in mitochondria (Chen et al., 2012). As one of the non-radical forms of ROS, H2O2 is able to inactivate enzymes by oxidizing their thiol groups causing oxidative stress (Gill and Tuteja, 2010). In the present study, low temperature stress increased H2O2 concentration in chloroplasts and mitochondria of wheat leaves, this could be related to the repressed capacity of sub-cellular oxygen scavenging systems under abiotic stresses (Gechev et al., 2006; Omoto et al., 2012). Most interestingly, NL plants had a higher H2O2 concentration in chloroplasts and mitochondria as compared with CL plants, indicating that cold priming enhanced the ROS scavenging capacity in wheat leaves. It is known that in the ROS scavenging systems, SOD catalyzes the disproportionation of singlet oxygen (1O2) generated from chloroplast and mitochondrial electron transport chains, and produces H2O2 (Keunen et al., 2013). Then CAT decomposes H2O2 to H2O and O2, together with APX, which play important roles in scavenging of ROS under normal and stress conditions (Keunen et al., 2013). Our previous and other studies have reported that earlier drought or heat priming can improve the antioxidant capacity in wheat leaves during subsequent stresses of high temperature (Wang et al., 2014), chilling (Thakur and Nayyar, 2013) and waterlogging (Li et al., 2011). In chloroplasts, the activities of SOD and APX were enhanced, while CAT activity was unaffected in CL plants in relation to NL plants for both cultivars. This indicated that the main defence capacity against chloroplastic ROS accumulation under low temperature stress in the cold-primed plants was attributed mainly to the cooperation of SOD and APX, the so-called “waterewater cycle” as described by Asada (1999). There is evidence that mitochondrial redox homeostasis is maintained with an activated oxygen scavenging system (Matamoros et al., 2013). The performances of antioxidant enzymes in mitochondria were different with those in chloroplasts in the present study. The activities of chloroplastic SOD and CAT were higher in the CL as compared with the NL plants, while no significant difference in APX activity among the treatments was found in Funo. In addition, higher activities of SOD and APX were found in the CL plants in HM18. This reveals that cold priming reduced the risk of low temperature induced membrane injury in chloroplasts and mitochondria with varied primary defense enzymes (Asada, 1999). In conclusion, earlier cold priming improved the tolerance of photosynthetic apparatus in winter wheat to later low temperature stress. This was mainly attributed to the activated antioxidant systems in chloroplasts and mitochondria during cold priming, which protected the energy transport in photosynthetic apparatus against the oxidative burst during subsequent low temperature stress. Acknowledgements This study is supported by projects of PAPD, the National Natural Science Foundation of China (31325020, 31028017, 31000686, 31171484), the Specialized Research Fund for the Doctoral Program of Higher Education (20120097110026), and the China Agriculture Research System (CARS-03). Author contribution D.J. and J.C. conceived the idea and led the study design. X.L. carried out the experiment, performed analyses and wrote the paper. F.L., T.D. and W.C. assisted with study design, data analysis and writing. All authors contributed to the editing of the manuscript.

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