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Plant Physiology and Biochemistry 112 (2017) 251e260

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Glycine increases cold tolerance in rice via the regulation of N uptake,physiological characteristics, and photosynthesis

Cao Xiaochuang a, 1, Zhong Chu a, 1, Zhu Lianfeng a, Zhang Junhua a, Sajid Hussain a,Wu Lianghuan b, Jin Qianyu a, *

a State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, 310006 Chinab Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, ZhejiangUniversity, Hangzhou, 310058 China

a r t i c l e i n f o

Article history:Received 22 November 2016Received in revised form10 January 2017Accepted 11 January 2017Available online 12 January 2017

Keywords:Cold stressNitrogen formAmino acidsPhotosynthesisPhysiological traitRice

Abbreviations: Fv/Fm, Maximum quantum yield ofefficiency of PSII; qP, Photochemical quenching; qN,ing; D, Antenna dissipative energy; P, Photochemistryenergy; DCD, Dicyandiamide; SOD, Superoxide dismPeroxidase; MDA, Malondialdehyde; AN, NH4

þ-N; NNControl temperature; L, Low temperature.* Corresponding author. State Key Laboratory of Ric

Research Institute, No. 359 Tiyuchang Road, HangzhouE-mail address: caoxiaochuang@126.com (J. Qiany

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.plaphy.2017.01.0080981-9428/© 2017 Elsevier Masson SAS. All rights res

a b s t r a c t

To investigate the response of rice growth and photosynthesis to different nitrogen (N) sources undercold stress, hydroponic cultivation of rice was done in greenhouse, with glycine, ammonium, and nitrateas the sole N sources. The results demonstrate that exposure to low temperature reduced the ricebiomass and leaf chlorophyll content, but their values in the glycine-treated plants were significantlyhigher than in the ammonium- and nitrate-treated plants. This might be attributed to the higher Nuptake rate and root area and activity in the glycine-treated plants. The glycine-treated plants alsomaintained high contents of soluble proteins, soluble sugars, and proline as well as enhanced antioxidantenzyme activities to protect themselves against chilling injury. Under cold stress, reduced stomatalconductance (gs) and effective quantum efficiency of PSII (FPSII) significantly inhibited the leaf photo-synthesis; however, glycine treatment alleviated these effects compared to the ammonium and nitratetreatments. The high non-photochemical quenching (qN) and excess energy dissipative energy (Ex) in theglycine-treated plants were beneficial for the release of extra energy, thereby, strengthening theirphotochemical efficiency. We, therefore, conclude that the strengthened cold tolerance of glycine-treatedrice plants was closely associated with the higher accumulation of dry matter and photosynthesisthrough the up-regulation of N-uptake, and increase in the content of osmoprotectants, activities of theantioxidant defense enzymes, and photochemical efficiency. The results of the present study providenew ideas for improving the plant tolerance to extreme temperatures by nutrient resource managementin the cold regions.

© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction

Plant species have evolved to function under optimal environ-mental conditions, for example, of light and temperature; de-viations in these conditions can result in the reduction of plant

PSII; FPSII, Effective quantumNon-photochemical quench-dissipative energy; Ex, Excessutase; CAT, Catalase; POD,, NO3

�-N; GN, Glycine-N; C,

e Biology, China National Rice, Zhejiang, 310006, PR China.u).

erved.

growth, inhibition of photosynthesis, imbalanced ion uptake, andoxidative stress (Sims et al., 2012; Genisel et al., 2013). Rice (Oryzasativa L.) is widely grown in tropical and subtropical areas. It facesunexpected chilling damage, especially in some areas of temperatezones with moderate climate (Oliver et al., 2005; Nagasuga et al.,2011). The unpredictable cold snaps generally cause an averageannual yield reduction of 5e10%, which could occasionally go up to20e40%, despite many efforts that have been made to developchilling-resistant cultivars (Oliver et al., 2005). Therefore,improving tolerance to extreme temperature through breeding orcultivation techniques is crucial to rice cultivation in the regionsthat experience extremely low temperatures.

During vegetative growth of rice, low temperature reduces thetillering rate, delays leaf development, and inhibits leaf elongation(Shimono et al., 2002). In some cases, these changes are accom-panied by yellowing of leaves, slow growth, delayed crop

C. Xiaochuang et al. / Plant Physiology and Biochemistry 112 (2017) 251e260252

maturation, and poor development (Suzuki et al., 2008). Lowtemperature also directly or indirectly impacts an array of photo-synthetic processes in chloroplasts, such as the thylakoid electrontransport, carbon reduction cycle, and control of stomatalconductance (Lambers et al., 2008). This leads to the generation ofreactive oxygen species (ROS) and further represses the netphotosynthetic rate because of the accumulation of photo-energy(Alam and Jacob, 2002). The accumulation of ROS has detrimentaleffects on plant growth because they are highly destructive tolipids, nucleic acids, and proteins. To protect against ROS, higherplant species generally utilize a defense system involving anti-oxidative compounds or enzymes (Apel and Hirt, 2004). Previousstudies have demonstrated that cold tolerant plants exhibit lowerreductions in chlorophyll content (Kuk et al., 2002), higher leafwater content (Kuk et al., 2002), and higher anti-oxidative enzymeactivity compared to the cold sensitive plants (Huang and Guo,2005). Besides, many other metabolites and plant growth regula-tors, e.g. melatonin, proline, and spermidine, also play a crucial rolein improving the resistance of plants to cold stress (Tan et al., 2012;Yamamoto et al., 2012).

Under low root-zone temperature, rice nitrogen (N) uptakeability is significantly reduced as a result of the reduced activity ofenzymes and transporters (Feng et al., 2011). Plant N preferentialuptake also varies with the soil or water temperature. Especially insome ecosystems with low temperature or lower N mineralizationrate, amino acid uptake even constitutes a high proportion of planttotal N economy compared to soil inorganic N (N€asholm et al.,2009). In addition, soil N forms have been demonstrated to beavailable for plant growth and photosynthesis, because of theirdifferent photo-energy consumption and reductant supply as wellas the different chlorophyll content, stomatal conductance andchloroplast volume (Guo et al., 2002, 2007; Liu et al., 2013). A majordifference in the soils under cold ecosystem, when compared tothose under the tropical ecosystems, is the change in the form ofavailable N; for example, the content of organic N increaseswhereas that of inorganic N decreases due to slow Nmineralizationrate. Plant photosynthesis is closely related to plant growth and drymatter accumulation. However, little is known, as of date, about theeffects of amino acid N derived from organic fertilizers, on ricegrowth and leaf photosynthesis under cold stress.

In this study, we determined whether amino acid derived N canimprove the cold tolerance of rice compared to the nitrate andammonium derived N, and further decipher the possible reasonsfor such tolerance. To evaluate this physiological response in rice, apot experiment was conducted under simulated cold stress con-dition in a green house, and the effects of different N forms on ricegrowth, N uptake, as well as on the physiological and photosyn-thetic traits during the vegetative period were examined underhydroponic cultivation with ammonium, nitrate and glycine astheir sole N nutrition.

2. Materials and methods

2.1. Plant materials, cultivation, and cold-stress treatment

Rice (Oryza sativa L. cv. ‘Zhongzheyou 1’ indica hybrid rice)seedlings were grown hydroponically in an environmentallycontrolled growth chamber. After germination on moist filter pa-per, rice seeds were transferred to a 2.0-mmol L�1 CaSO4 solutionfor germination. Three days later, rice seedlings were transferred toblack plastic pots containing 1/4th -strength mixture of NO3

� andNH4

þ nutrient solution (for composition, see below). Ninety potswith internal radius of 6.0 cm and total depth of 12 cmwere used inthe experiment. Each pot was covered with a plastic cap, which hada 2.5-cm diameter hole drilled in the center for seedling to grow out

of the pot. After three days in the pot, the seedlings were suppliedwith half-strength mixture of the nutrient solution. Subsequently,after three more days, the rice seedlings were supplied with full-strength nutrient solution for one week. The composition of thenutrient solution for hydroponic culture was as follows: macro-nutrients (mmol L�1): 2.85 N as (NH4)2SO4þCa(NO3)2; 1.02 K asK2SO4 and KH2PO4; 0.32 P as KH2PO4, 1.65 Mg as MgSO4; micro-nutrients (mmol L�1): 35.8 Fe as Fe-EDTA; 9.10 Mn as MnSO4; 0.15Zn as ZnSO4; 0.16 Cu as CuSO4; 18.5 B as H3BO3; 0.52 Mo as(NH4)6Mo7O24; 0.1 Si as Na2SiO4 (Li et al., 2012). Dicyandiamide(DCD, 0.2 mg L�1) was added to each pot as the nitrification in-hibitor. The solution pH was maintained at 5.50 ± 0.05 with the1 mol L�1 HCl or 1 mol L�1 NaOH.

One week later, the seedlings were supplied with the nutrientsolution containing either 2.85 mmol L�1 NH4

þ-N (AN),2.85 mmol L�1 NO3

�-N (NN), or 2.85 mmol L�1 glycine-N (GN) astheir sole N source, and others elements were provided asmentioned above. The treatments with each N source were done in30 replicates. The seedlings from each N treatment were dividedinto two groups: seedlings in one group were grown in a PGW36controlled environment growth chamber at 28 �C during daytimeand 20 �C during night (the control temperature treatment, C),whereas seedlings in the other group were grown at 18 �C duringdaytime and 10 �C during night (the low temperature treatment, L).The six treatments groups were abbreviated as follows: NH4

þ-N atcontrol temperature (AN-C), NH4

þ-N at low temperature (AN-L),NO3

�-N at control temperature (NN-C), NO3�-N at low temperature

(NN-L), glycine-N at control temperature (GN-C), and glycine-N atlow temperature (GN-L). In ammonium-containing nutrient solu-tion, Ca2þ was supplied as CaCl2 (1.43 mmol L�1). In addition, in theglycine treatments, the nutrient solutions contained 10 mg ampi-cillin L�1 to prevent the rapid de-amination of glycine, whereas inthe ammonium and nitrate treatments, they contained 0.2 mg L�1

dicyandiamide (DCD). Each treatment had 15 replicates arranged ina completely randomized design (CRD) to avoid edge effect in thechamber. The nutrient solutions were changed every three days.The chamber wasmaintainedwith a 12-h photoperiod, 60% relativehumidity, and a photosynthetic photon flux density (PPFD) of1000 mmol m�2 s�1 during the photoperiod.

2.2. Gas-exchange and fluorescence measurement

Two weeks after the start of cold stress (35 days after sowing),light-saturated photosynthesis of the fourth newly expanded leaf ineach treatment was measured from 09:00 to 15:00 h using a Li-Cor6400 portable photosynthesis open system (Li-Cor Co. Ltd. UAS) inthe temperature-controlled chamber. The leaf temperature duringthe measurements was maintained at 30 �C, with a PPFD of1500 mmol m�2 s�1. The ambient CO2 concentration in the cham-ber was adjusted to 380 mmol mol�1 with a CO2 mixture, and therelative humidity was maintained at 1.4e1.6 kPa. Data wererecorded after equilibration to a steady state. The gas-exchange andfluorescence parameters were measured in five replicates for eachtreatment. These leaves were labeled, and the fluorescence mea-surements mentioned below were also made on the same leaves.

One day later, measurement of chlorophyll fluorescence wasconducted on the above-mentioned labeled leaves with a portablepulse amplitude modulation fluorimeter (PAM-2000; Heinz WalzGmbH, Effeltrich, Germany). The chlorophyll fluorescence of thelabeled leaf in the dark was monitored for approximately 30 min at25 �C with an 800 ms pulse (8000 mmol m�2 s�1, 20 KHz) of satu-rating light, and the maximum quantum yield of PSII (Fv/Fm,Fv ¼ Fm-Fo) was determined after the fluorescence had reached asteady level. Fm and Fo represent the maximal and minimal fluo-rescence in the absence of non-photochemical quenching (dark-

C. Xiaochuang et al. / Plant Physiology and Biochemistry 112 (2017) 251e260 253

adapted stage). After determination of Fv/Fm, the actinic light(500 mmol photons m�2 s�1) was supplied for 15 min. Fm’ (maximalfluorescence in the presence of non-photochemical quenchingduring illumination), Fs (steady-state fluorescence) and Fo’ (mini-mal fluorescence in the presence of non-photochemical quenchingduring illumination) were determined to calculate the other chlo-rophyll fluorescence parameters according to themethod describedby Demmig-Adams et al. (1996). The effective quantum efficiency ofPSII (FPSII) was defined as (Fm’-Fs)/Fm’. The quenching coefficient forphotochemical fluorescence quenching (qP) was calculated as (Fm’-Fs)/(Fm’-Fo’), whereas the quenching coefficient for non-photochemical fluorescence quenching (qN) was calculated as(Fm/Fm’-1).

As described by Demmig-Adams et al. (1996), the light energyutilized by plants can be divided into three parts: (1) photochem-istry dissipative energy (P), which is the fraction of light absorbedin PSII antennae that is utilized in photosynthetic electron trans-port, (2) antenna pigment dissipative energy (D), which is thefraction of light absorbed in PSII antennae that is dissipated viathermal energy dissipation in antennae, (3) excess energy (Ex),which is a parameter reflecting the excess light energy that is notused for photosynthetic electron transport or non-photochemicaldissipation, correspondingly calculated as follows:

P ¼ Fv’=Fm’,qP;D ¼ 1� Fv’=Fm’;

Ex ¼ ðFv’=Fm’Þ,ð1� qPÞ

2.3. Root 15N uptake rate

After the gas-exchange and fluorescence measurements, thesame five replicates of each treatment were used for the mea-surement of root N uptake rate. Firstly, the rice seedlings weretreated with deionized water for 4 h to create a nutrient starvationcondition. Thereafter, root N uptake rates at each control and lowtemperature were determined from the uptake of the2.85 mmol L�1 15N labeled substrates (50 atom% 15NO3

�, 50 atom%15NH4

þ, 50 atom% 15N-glycine; all purchased from ShanghaiResearch Institute of Chemical Industry, China). In addition to N, theuptake solutions also contained the aforementioned macronutri-ents and micronutrients as described in 2.1. The uptake solution forthe glycine treatments contained 10 mg L�1 ampicillin, whereasthose for the ammonium and nitrate treatments contained0.2 mg L�1 DCD. The rice seedlings were cultivated under condi-tions of 12-h photoperiod, 60% relative humidity, and a PPFD of1500 mmol quanta m�2 s�1 during the photoperiod. For controltemperature treatments, the temperature was maintained at 28 �Cduring daytime and 20 �C during night, whereas for low temper-ature treatments, it was kept at 18 �C during daytime and 10 �Cduring night, as described above. After 6 h of incubation, the rootsystem was cut off, washed with 50 mmol L�1 CaCl2, and rinsedwith deionized water. The leaf area was determined by a Li-Cor3100C leaf area meter (Li-Cor, USA) and the root morphology wasvisualized after scanning with an Epson Expression 10000XLscanner (Seiko Epson, Japan). Then rice shoots (leaves plus stems)and roots were stored at -80 �C, freeze-dried (Labconco FreezenSystem, USA), and their total biomass was determined byweighing.Thereafter, the harvested plant materials were ground to finepowder with a ball mill (Retsch MM301, German). 15N enrichmentin the dried roots and N content in the leaves were determinedusing a Tracer MAT-271 (Finnigan MAT, USA).

2.4. Physiological index measurement

All the newly expanded leaves of the rice seedlings from theremaining ten replicates for each treatment were sampled, frozenin liquid nitrogen, and then stored at -80 �C. The different physio-logical parameters for these leaves were determined, as mentionedbelow: leaf carotenoid, chlorophyll a, and chlorophyll b contentswere determined after 80% acetone soaking extraction, as describedby Li et al. (2012), and subsequent measurement of the absorbanceusing a Power WaveX microplate spectrophotometer (Bio-Tek In-struments, Inc. Winooski, VT, USA) at 480, 648, and 664 nm,respectively. The total chlorophyll content was calculated by addingthe values of chlorophyll a, chlorophyll b, and carotenoid contents.

Leaf proline content was estimated using the techniquedescribed as follow: the leaf samples (0.5 g) were homogenized in7.5 mL of 3% sulfosalicylic acid and the homogenate was filtered.Two milliliter of filtrate was mixed with 2 mL of ninhydrin reagentand 2 mL of glacial acetic acid. The reaction mixture was heated at100 �C for 1 h and then placed on ice for 20 min before extractionwith 4 mL of toluene. The absorbance of the chromospheres in thetoluene fraction was measured at 520 nm and the amount of pro-line was determined by comparison with a standard curve.

The soluble protein content was measured according to themodified method of Xu et al. (2015) and Turk et al. (2014). The leafsamples (0.5 g) were ground and extracted in a sodium phosphatebuffer (50 mmol L�1, pH 7.0). The extracts were centrifuged(4000 � g, 10 min at 4 �C) and the soluble proteins in the super-natant were quantified using bovine serum albumin as a standard.The soluble sugar was quantified using the anthrone colorimetrymethod.

The plasma membrane damage was evaluated using the pro-tocol for the measurement of percentage of electrolyte leakage.Approximately 0.3 g samples of fresh leaves were cut into 1-cmpieces and placed in test tubes containing 30 mL of deionizedwater. Subsequently, the tubes were incubated in a 30 �C waterbath for 2 h, after which the initial electrical conductivity (EC1) ofthe medium was measured. The samples were then placed in aboiling water bath at 95 �C for 15 min to release all the electrolytes.After the samples were cooled, their final electrical conductivity(EC2) was determined. The electrolyte percentage was calculatedusing the following formula:

½EC1=EC2� � 100

2.5. Measurement of enzyme activities

For estimating the activities of the antioxidant enzymes, leafsamples (0.5 g) were homogenized in 5 mL of 10 mmol L�1 phos-phate buffer (pH 7.0) containing 4% (w/v) polyvinylpyrolidone and1 mmol L�1 ethylenediaminetetraacetic acid. The homogenate wascentrifuged at 12,000 � g for 15 min at 4 �C and the supernatantwas used as the source of enzymes for estimation. The extractionwas carried out at 4 �C. The superoxide dismutase (SOD) activitywas estimated according to the method of Agarwal and Pandey(2004). One unit of SOD activity was defined as the amount ofenzyme that would inhibit 50% photoreduction of nitroblue tetra-zolium chloride.

The catalase (CAT) activity was determined using the methoddescribed by Turk et al. (2014) by monitoring the disappearance ofH2O2 at 240 nm. One unit of CATactivity was defined as the amountof enzyme required to decompose 1 mmol of H2O2 per minute at

C. Xiaochuang et al. / Plant Physiology and Biochemistry 112 (2017) 251e260254

25 �C.The malondialdehyde (MDA) content was measured according

to Velikova et al. (2000) using the thiobarbituric acid (TBA) test thatdetermines MDA as an end product of lipid peroxidation. Theamount of MDAeTBA complex was calculated from the extinctioncoefficient 155 (mmol l�1)�1 cm�1.

The activity of guaiacol oxidase (POD) was assayed using themethod of Sachadyn-Kr�ol et al. (2016). The reaction mixture con-tained 0.5 mL of 20 mmol L�1 guaiacol, 0.95 mL of 50 mmol L�1

acetic buffer (pH 5.6), and 0.05 mL of the enzyme extract. The re-action was started by adding 0.5 mL of 60 mmol L�1 H2O2. Theincrease in absorbance was spectrophotometrically measured at470 nm for 1 min. The POD activity was expressed as

DA470min�1g�1FM

Fig. 1. Rice leaf Chl-a, Chl-b, carotenoid contents of the three different N forms underthe control temperature (C) and low temperature (L). AN, NN and GN-L represent theNH4

þ-N, NO3�-N and glycine-N, respectively. The letters indicate the significant differ-

ence of rice total chlorophyll content among the six different treatments (p < 0.05). TheN, T and N � T represent the N main effect, temperature main effect and its interactioneffect, respectively (*p < 0.05; **p<0.01; nsp >0.05, non significant). The same as below.

2.6. Statistical analysis

All statistics, including analysis of variance (ANOVA), interactioneffect and least significant means, were performed using the SPSSsystem for Windows, version 14.0 (SPSS Inc. USA). Duncan's leastsignificant difference test was used to distinguish among the in-dividual mean values where applicable with a confidence level ofp < 0.05.

3. Results

3.1. Agronomic characters and rice biomass

N form and temperature had a significant effects on rice agro-nomic characters, including height, leaf area and biomass (p < 0.05,Table 1). Under the control temperature, rice leaf area, shootbiomass, and total biomass in GN-C were significantly higher thanin AN-C and NN-C (Table 1, p < 0.05). Compared to the controltemperature, cold stress significantly decreased the height, leafarea, shoot biomass, root biomass (except for the root biomass inAN-L), and total biomass of rice in the three different N forms, butshoot biomass and total biomass in GN-L were significantly higherthan in AN-L and NN-L (p < 0.05). In contrast, rice tiller number androot/shoot ratio in NO3

�, NH4þ, and glycine treatments, and SLN

(specific leaf N content, leaf N content/leaf area) in NO3� and NH4

þ

Table 1The agronomic characteristics of rice seedlings under the control temperature (C) and lo

Treatmentsb Height Tillers No., Leaf area Shoo

N form(N) Temperature(T) cm / cm2 g pla

AN C 67.7 ± 1.2aa 4.8 ± 0.4b 224.2 ± 16.9bc 1.1 ±L 47.8 ± 0.3bc 5.6 ± 0.2ab 160.0 ± 9.6d 0.9 ±

NN C 66.1 ± 0.7a 6.3 ± 0.6a 252.3 ± 10.3b 1.1 ±L 40.8 ± 8.1c 6.4 ± 0.7a 179.6 ± 6.5cd 0.9 ±

GN C 69.1 ± 1.9a 6.3 ± 0.3a 320.7 ± 11.7a 1.4 ±L 51.1 ± 1.4b 6.8 ± 0.2a 161.2 ± 13.4d 1.0 ±

Average AN 57.8 ± 0.7ab 5.3 ± 0.3b 192.2 ± 22.3a 1.0 ±NN 53.4 ± 4.4b 6.3 ± 0.6a 215.9 ± 21.2a 1.0 ±GN 60.1 ± 1.6a 6.5 ± 0.3a 241.2 ± 20.4a 1.2 ±

Average C 67.6 ± 1.3a 5.8 ± 0.4a 261.9 ± 24.3a 1.2 ±L 46.6 ± 3.3b 6.3 ± 0.4a 167.6 ± 14.4b 1.0 ±

F value N 57.2* 5.7* 10.2** 6.6**

T 86.3** 2.2 ns 121.8** 20.8N � T 28.6* 3.5* 10.7** 1.2ns

a Values followed by different letters within the same column are significantly differeb AN, NN and GN represent the ammonium, nitrate and glycine, respectively; C and L r

leaf N content: leaf area. The same as below.

treatments, showed no significant differences between the low andcontrol temperatures.

3.2. Leaf chlorophyll and N contents

The N form, temperature, and their interaction significantlyaffected leaf total chlorophyll, leaf N and root N contents (p < 0.05,Figs. 1 and 2). Specially, leaf total chlorophyll content in the leavesin AN-C group was significant higher than in NN-C and GN-C (Fig. 1,p < 0.05). The cold stress decreased the contents of total chloro-phyll, leaf N, and root N (except the root N content in NN-L group)for the three N forms (Figs. 1 and 2). However, the total chlorophyllcontent in the leaves in GN-L groupwas significantly greater than inAN-L and NN-L groups (p < 0.05). However, leaf Chl-a, Chl-b, andcarotenoid contents showed no significant difference between GN-L and GN-C groups (p > 0.05).

w temperature (L).

t biomass Root biomass Total biomass Root/Shoot ratio SLN

nt�1 g plant�1 g plant�1 / mg cm�2

0.08b 0.18 ± 0.01bc 1.33 ± 0.05b 6.0 ± 0.1ab 0.14 ± 0.01b0.07c 0.15 ± 0.01c 1.06 ± 0.11c 6.1 ± 0.4ab 0.18 ± 0.04ab0.05b 0.21 ± 0.01ab 1.27 ± 0.09b 5.3 ± 0.2c 0.16 ± 0.03ab0.1c 0.16 ± 0.02c 1.08 ± 0.07c 5.5 ± 0.4bc 0.16 ± 0.02ab0.03a 0.24 ± 0.01a 1.65 ± 0.04a 6.0 ± 0.3ab 0.14 ± 0.02b0.05b 0.17 ± 0.01c 1.24 ± 0.05b 6.3 ± 0.4 a 0.19 ± 0.02a0.07b 0.17 ± 0.04a 1.20 ± 0.08ab 6.0 ± 0.4ab 0.16 ± 0.03a0.06b 0.19 ± 0.03a 1.17 ± 0.070b 5.4 ± 0.3b 0.15 ± 0.02a0.05a 0.20 ± 0.04a 1.40 ± 0.05a 6.2 ± 0.5a 0.17 ± 0.02a0.12a 0.21 ± 0.03a 1.40 ± 0.11a 5.8 ± 0.5a 0.15 ± 0.02a0.09b 0.16 ± 0.03b 1.11 ± 0.09b 6.0 ± 0.4a 0.18 ± 0.03a

4.1* 6.4** 3.0 ns 0.2 ns

** 23.6** 22.9** 0.5 ns 3.5 ns

1.7ns 1.4ns 0.1 ns 1.5 ns

nt (p < 0.05) according to Duncan's multiple range test. The same as below.epresent the control temperature and low temperature; SLN represents the ratio of

Fig. 2. Rice root N (a) and shoot N (b) contents of the three different N forms under thecontrol temperature (C) and low temperature (L).

Fig. 3. Root NO3�, NH4

þ and glycine uptake rate by rice seedlings under the controltemperature (C) and low temperature (L).

C. Xiaochuang et al. / Plant Physiology and Biochemistry 112 (2017) 251e260 255

3.3. Root 15N uptake rate and morphological traits

Under the control temperature, the root 15N uptake rate in GN-Cwas significant higher than in AN-C and NN-C (p < 0.05, Fig. 3).Compared to the control temperature, cold stress significantlyreduced root the 15N uptake rate and the root morphology traits(e.g., root length, area, volume, and root activity) in the threedifferent N forms (Fig. 3, Table 2). However, the 15N uptake rate in

GN-L was significant higher than that in AN-L under the low tem-perature (p < 0.05), which was consistent with the trend of varia-tion in the root area and root activity.

3.4. Physiological characteristics and enzymatic activity

Analysis of variance indicated significant effects of N form, andtemperature as well as their interactions on the different physio-logical characteristics (except soluble protein) and enzymatic ac-tivity (p < 0.05, Figs. 4 and 5). Compared to the control temperature,cold stress significantly increased the contents of leaf soluble sugar,soluble protein, and proline in GN -L (p < 0.05, Fig. 4), but theirsoluble sugar and soluble protein contents showed no significantdifferences between the cold stress and control temperature in ANand NN treatments. In contrast, the cold stress significantlyincreased the leaf electric conductivity in AN-L and NN-L under thelow temperature, but its value in GN groups showed no significantdifference between the control and low temperatures.

Under the control temperature, the leaf POD and SOD activities,and the MDA content (except in NN-C) showed no significant dif-ferences among the three different N forms (p > 0.05, Fig. 5). Inaddition to the CAT activity in GN-L, the cold stress significantlyincreased the leaf CAT, POD, and SOD activities, and the MDAcontent in GN-L, AN-L, and NN-L groups, when compared to theactivities under the control temperature. Especially, the POD andSOD activities in GN-L were significant higher than in AN-L and NN-L under the low temperature, whereas the MDA content showedthe opposite trend (p < 0.05).

3.5. Leaf photosynthetic parameters

Temperature had a significant effect on leaf net photosyntheticrate (Pn) and the different chlorophyll fluorescence parameters,while N forms showed the opposite trends (Tables 3 and 4).Specially, the Pn, FPSII, qP, qN, D, P, and Ex of the rice leaves, showedno significant differences among the GN-C, AN-C, and NN-C groups(p > 0.05, Tables 3 and 4). Compared to the control temperature,cold stress significantly reduced the leaf Pn, Gs, Tr, FPSII, qP, P, and Ex(p< 0.05), but increased the qN and D in AN-L and NN-L treatments.The values of leaf Pn, Gs, and Ci and the aforementioned chlorophyllfluorescence parameters (including FPSII, qP, D, P, and Ex) in GN-Lwere not significantly different from those in GN-C, irrespectiveof the cold stress, and specially, the values of Pn,FPSII, and Ex in GN-Lwere significant higher than those in AN-L and NN-L under the lowtemperature.

4. Discussion

4.1. Rice growth and root morphological characters under coldstress

Plant growth inhibition under chilling stress, caused by areduction in leaf area, nutrient uptake, cytoskeleton, photosyn-thesis, enzyme activity, membrane fluidity, etc. Has been describedin several reports (Shimono et al., 2004; Ploschuk et al., 2014).Shimono et al. (2002) reported that the dry matter production ofrice grown in low flooded-water temperature decreased severely,with decreased tiller number and leaf number during the vegeta-tive period. In our study, obvious differences in chilling sensitivitywere found in rice seedlings grown under the three different Nforms (Table 1). The cold stress significantly decreased the rice leafarea and biomass (except the root biomass in AN-L) in the NO3

�,NH4

þ, and glycine treatments. The leaf chloroplasts are the mainplace for photosynthesis. This change indicates that the inhibitionof allocation of photosynthates to the aboveground part may be

Table 2Rice root morphology characteristics and root activity under the control temperature (C) and low temperature (L).

Treatments Length Diameter Surface area Volume Tips Activity

N form(N) Temperature(T) cm mm cm2 cm3 / mg g�1 h�1

AN C 976.9 ± 32b 0.41 ± 0.01ab 126.5 ± 4.8b 1.58 ± 0.06ab 2378.5 ± 114.6ab 216.4 ± 23.1abL 622.6 ± 41.7c 0.39 ± 0.01b 76.8 ± 5.3d 0.95 ± 0.08c 1974.2 ± 102.4bc 152.3 ± 8.8d

NN C 1141.4 ± 44.1a 0.44 ± 0.01a 137.4 ± 7.2ab 1.30 ± 0.06b 2571.4 ± 122.4a 197.1 ± 27.3bcL 653.3 ± 48.5c 0.43 ± 0.001a 88.2 ± 7.4d 0.75 ± 0.05c 1742.2 ± 69.6c 160.1 ± 15.9d

GN C 1009.1 ± 65.1ab 0.45 ± 0.01a 141.2 ± 6.6a 1.72 ± 0.11a 2552.2 ± 168.6a 229.3 ± 14.9aL 628.8 ± 26.5c 0.42 ± 0.01ab 108.6 ± 3.9c 0.86 ± 0.05c 2108.2 ± 157.6b 186.9 ± 12.1c

Average AN 799.8 ± 82.4a 0.40 ± 0.01a 101.7 ± 11.3b 1.27 ± 0.22a 2176.9 ± 107.7a 184.6 ± 16.1bNN 902.3 ± 103.5a 0.43 ± 0.03a 112.8 ± 10.9ab 1.03 ± 0.12b 2156.8 ± 96.0a 178.8 ± 16.7bGN 819.0 ± 102.1a 0.43 ± 0.04a 124.9 ± 5.8a 1.30 ± 0.12ab 2330.2 ± 163a 208.1 ± 13.6a

Average C 1042.8 ± 48.2a 0.43 ± 0.04a 135.7 ± 6.5a 1.53 ± 0.24a 2500.7 ± 56.8a 214.5 ± 21.8aL 634.9 ± 38.9b 0.41 ± 0.01a 92.5 ± 5.4b 0.86 ± 0.15b 1939.2 ± 111.1b 166.4 ± 12.4b

F value N 3.0ns 6.4ns 1.2ns 9.2** 3.2ns 20.3*

T 126.7** 149.2** 3.6ns 4.5** 22.2** 18.6**

N � T 1.5 ns 1.4ns 1.2ns 1.3ns 1.4ns 4.7ns

Fig. 4. Electric conductivity (a), soluble sugar (b), soluble protein (c) and proline (d) contents in leaves of rice seedling under the control temperature (C) and low temperature (L).

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attributed to the reduction of leaf area. This may also be attributedto the decrease in turgidity of the cells and the consequentshrinkage of cells, which correspondingly reduce the leaf area inorder to alleviate the cold injury (Erdal, 2012). This precaution isone of the first responses given by plants to cope with the coldstress (Nayyar et al., 2005). The present study demonstrates thatthe cold-induced decreases in shoot biomass and total biomasswere ameliorated by exogenous glycine application, which dem-onstrates that glycine application can partly reduce the negativeeffects of cold stress compared to the inorganic N. Compared to the

ammonium and nitrate treatments, the increased root/shoot ratioin the glycine treatment was also beneficial for increase in thevalues of root morphology traits and the formation of the above-ground part, thus, alleviated the cold damage (Table 1).

Because plants acquirewater and nutrients mainly through root,any plasticity in root architecture enables the plants to respond to achanging environment (Xu et al., 2015). Nagasuga et al. (2011)demonstrated that dry matter production under low root temper-ature was controlled mainly by the quantitative growth parame-ters, such as leaf area, root area, and activity. Under low root-zone

Fig. 5. Activities of CAT (a), POD (b), SOD(c) and MDA (d) in leaves of rice seedlings under the control temperature (C) and low temperature (L).

Table 3Photosynthesis (Pn), stomatal conductance of CO2 (gs), intercellular CO2 concentration (Ci) and Transpiration rate (Tr) in leaves of rice seedlings under the control temperature(C) and low temperature (L).

Treatments Pn gs Ci Tr

N form(N) Temperature(T) mmol m�2 s�1 mol m�2 s�1 mmol mol�1 mmol m�2 s�1

AN C 23.7 ± 0.8a 0.21 ± 0.02a 224.7 ± 6.5ab 4.7 ± 0.3bL 19.2 ± 0.5c 0.15 ± 0.01c 214.7 ± 4.0bc 3.4 ± 0.1c

NN C 24.0 ± 0.7a 0.17 ± 0.01bc 237.4 ± 6.2a 5.2 ± 0.1aL 17.6 ± 0.7c 0.09 ± 0.01d 212.6 ± 7.1bc 3.6 ± 0.2c

GN C 22.6 ± 0.7ab 0.19 ± 0.01ab 210.7 ± 3.6bc 4.3 ± 0.1bL 21.5 ± 0.7b 0.17 ± 0.01bc 202.9 ± 5.6c 3.6 ± 0.02c

Average AN 21.4 ± 2.7a 0.18 ± 0.02a 219.7 ± 9.1ab 4.0 ± 0.3aNN 20.8 ± 1.0a 0.13 ± 0.02b 225.0 ± 11.5a 4.4 ± 0.3aGN 22.0 ± 1.2a 0.18 ± 0.01a 206.8 ± 8.4b 3.9 ± 0.4a

Average C 23.4 ± 1.2ab 0.19 ± 0.02a 224.2 ± 14.3a 4.7 ± 0.5aL 19.4 ± 2.0b 0.14 ± 0.03b 210.0 ± 10.1a 3.5 ± 0.2b

F Value N 1.7ns 16.9** 5.5* 4.8*

T 57.1** 46.0** 9.5** 87.1**

N � T 8.6* 5.9* 1.3ns 3.7ns

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temperature, rice root N uptake ability was significantly reduceddue to the reduction of root activity (Feng et al., 2011), which wasconsistent with our results. However, under glycine treatment, theN uptake rate was higher compared to that in the ammoniumtreatment, irrespective of the cold stress. This higher uptake rateunder glycine treatmentmay be attributed to the high root area androot activity, which are beneficial for the increase of the total Ncontent compared to that under the ammonium treatment. Other

studies have demonstrated that plant growth and the growth pa-rameters were closely related to plant photosynthesis (Guo et al.,2007). It is pertinent to determine if there is any beneficial effectof glycine nutrition on the photosynthetic process in rice, and todecipher the mechanism by which glycine nutrition alleviates thecold injury.

Table 4Chlorophyll fluorescence parameters in leaves of rice seedlings under the control temperature (C) and low temperature (L).

Treatments Fv/Fma FPSII qP qN D P Ex

N form(N) Temperature(T)

AN C 0.672 ± 0.051a 0.217 ± 0.003ab 0.520± 0.035a 0.513± 0.026c 0.553 ± 0.026bc 0.230 ± 0.029ab 0.217 ± 0.003aL 0.427 ± 0.039c 0.190 ± 0.003c 0.393 ± 0.020bc 0.677 ± 0.032ab 0.707 ± 0.019a 0.117 ± 0.012c 0.177 ± 0.007b

NN C 0.628 ± 0.074a 0.223 ± 0.004a 0.553 ± 0.038a 0.493 ± 0.043c 0.517 ± 0.043c 0.270 ± 0.044a 0.213 ± 0.003aL 0.486 ± 0.065bc 0.187 ± 0.003c 0.343 ± 0.032c 0.69 ± 0.040ab 0.727 ± 0.020a 0.093 ± 0.015c 0.181 ± 0.006b

GN C 0.554 ± 0.064b 0.210 ± 0.006ab 0.493 ± 0.043ab 0.583 ± 0.038bc 0.563 ± 0.033bc 0.220 ± 0.038ab 0.220 ± 0.006aL 0.512 ± 0.061bc 0.203 ± 0.003b 0.413 ± 0.024bc 0.710 ± 0.046a 0.647 ± 0.026ab 0.147 ± 0.018bc 0.207 ± 0.009a

Average AN 0.550 ± 0.051a 0.204 ± 0.004a 0.467 ± 0.046a 0.605 ± 0.039a 0.635 ± 0.027a 0.173 ± 0.021a 0.197 ± 0.008aNN 0.558 ± 0.072a 0.205 ± 0.003a 0.457 ± 0.038a 0.596 ± 0.047a 0.624 ± 0.031a 0.182 ± 0.030a 0.197 ± 0.007aGN 0.533 ± 0.063a 0.207 ± 0.005a 0.452 ± 0.036a 0.654 ± 0.042a 0.605 ± 0.038a 0.184 ± 0.028a 0.213 ± 0.012a

Average C 0.618 ± 0.062a 0.217 ± 0.005a 0.526 ± 0.041a 0.533 ± 0.045b 0.545 ± 0.036b 0.244 ± 0.026a 0.221 ± 0.018aL 0.475 ± 0.054b 0.194 ± 0.003b 0.387 ± 0.052b 0.695 ± 0.051a 0.694 ± 0.041a 0.123 ± 0.021b 0.192 ± 0.016a

F value N 0.2ns 0.08ns 0.6ns 1.3ns 0.4ns 0.07ns 5.3*

T 16.3* 1.2ns 26.8** 27.1** 39.4** 27.3** 35.6ns

N � T 1.6ns 2.5ns 2.0ns 0.4ns 2.4ns 1.7ns 2.7ns

a FPSII, qP, qN, D, P, Ex represent the (Fm' -Fs)/Fm' , photochemical quenching, non-photochemical quenching, antenna dissipative energy, photochemistry dissipative energyand non-photochemistry dissipative energy, respectively.

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4.2. Enhanced growth of glycine-treated plants was related tohigher content of osmoprotectant and strengthened antioxidantdefense system

The chilling stress significantly damages the photosyntheticprocesses in the chloroplasts, leading to the generation of ROS bysurplus photo-energy (Alam and Jacob, 2002). The accumulation ofROS leads tomembrane damage, denaturation of biomolecules, andcell death. This was confirmed by the high cell permeability (highelectric conductivity) induced by cold stress in our study; its valuein GN-L treatment was obviously lower than in AN-L and NN-L(Fig. 4a). To protect against ROS, higher plants generally developother protection mechanisms to accumulate various osmopro-tectants, such as soluble proteins, carbohydrates, and glycinebetaine (Apel and Hirt, 2004; Faize et al., 2011). The presentinvestigation showed that the soluble protein and soluble sugarcontents in ammonium and glycine treatments were greatlyincreased under the cold stress conditions when compared to theircontents under the control temperature conditions, especially inthe glycine treatment group (Fig. 4b and c). Previous reports indi-cate that proline plays an important role in the osmotic adjustment,scavenging of ROS, protection of membrane integrity, and increaseof enzyme activities (Nayyar et al., 2005; Ashraf and Foolad, 2007).Correspondingly, in the present study, we observed high prolinecontent in the glycine-treated rice plants, which also enhanced thecold tolerance of the plants. C and N are essential for plant toperform their routine and fundamental cellular activities duringdevelopment, N metabolism regulation is crucial for stress toler-ance. Lawlor reported that the interference between stress and Nassimilation is a complex network that influences almost allphysiological processes in plants (2002). NO3

�, after being taken upinto the cell, is reduced to NH4

þ by NR and NiR, and then translatedinto the amino acids by the GS, GOGAT and some transaminaseenzymes (such as GPT, GOT) (Kaiser and Huber, 2001). These pro-cesses require energy and C skeleton, which are provided byvarious carbohydrates and their metabolic products (Britto andKronzucker, 2002). Wang et al. (2009) demonstrated that gluta-mine absorbed into plant were not converted into NO3

� in tissuespart. So we hypothesized that the utilization of the small moleculeorganic matter (e.g. glycine) may be more efficiency compared tonitrate and ammonium, and the absorbed glycine may be imme-diately assimilated in root via the process of transamination anddeamination, then up-translated to the protein and others aminoacids in the above-ground part (such as proline). In addition, underthe cold stress conditions, the higher uptake rate of glycine, also

accelerated the upward transportation of the assimilation product.Cao revealed that the high uptake rate of glycine may be closelyrelated to its high maximal velocity (Vmax) and low affinity constant(Km) compared to the nitrate treatment (Cao et al., 2015). In a word,the high utilized efficiency and uptake rate for glycine partlyexplain the high contents of proline, carbohydrates and solubleproteins in the glycine treatment under the cold stress condition.

In addition to increasing the content of osmoprotectants, plantsdevelop other strategies to protect themselves against harmful ef-fects of ROS. The most important of these strategies, is to boost theantioxidant defense system, consisting of enzymatic and non-enzymatic components, such as SOD, glutathione peroxidase(GPX), CAT, and ascorbate peroxidase (APX) (Benavides et al., 2000;Shao et al., 2008). High MDA content is presumably associated withthe elevated ROS production, which occurs as a consequence ofcold-induced electrolyte leakage from the electron transport sys-tem in photosynthesis and respiration (Turk et al., 2014). Comparedto the ammonium and nitrate treatments, the treatment withglycine markedly reduced the increase in the level of MDA, andsignificantly increased the activities of POD and SOD under coldstress. The present results were partly consistent with previousfindings that the application of organic matter stimulated the ac-tivities of the antioxidant enzymes, such as SOD, APX, and GR (Tanet al., 2012). However, unlike the variations in the activities of PODand SOD, the CATactivity showed no significant difference betweenthe two GN groups, irrespective of the cold stress or not; this shoulddeserve more attention in future research.

4.3. Glycine increased the rice leaf photosynthesis via the up-regulation of chlorophyll content, FPSII, and Ex under cold stress

Photosynthesis is the most temperature-sensitive metabolicprocess in higher plants (Lambers et al., 2008). Excessive ROSproduced by cold stress causes serious structural and functionaldamage, including overoxidation of thylakoid membranes, pri-marily in the chloroplasts. Some authors have generally attributedthe pigment loss to the destruction of chlorophyll structure byexcessive ROS and/or by the stimulation of chlorophyllase activity(Triantaphylides and Havaux, 2009). The efficiency of photosyn-thesis, is, therefore, reduced significantly (Tan et al., 2012). In ourstudy, the chlorophyll contents in AN-L and NN-L were significantlyreduced compared to the control temperature. However, the chlo-rophyll contents in GN-L were not significantly different from thatin GN-C. It indicates that the exogenous glycine application cangreatly alleviate the reduction in the pigment content relative to

C. Xiaochuang et al. / Plant Physiology and Biochemistry 112 (2017) 251e260 259

ammonium and nitrate applications. As mentioned above, thismitigating effect of glycine might be attributed to the decreasedlevels of ROS as a consequence of its direct antioxidant character-istic and stimulating effect on the antioxidant system.

Exposure to low temperature leads to decreased CO2 assimila-tion, which can be attributed to the closure of stomata, photo-inhibition, changes in the transcript levels and expression ofphotosynthesis related enzymes, end-product limitation, and othernon-stomatal factors (Allen and Ort, 2001; Yamori et al., 2012). Inaddition, N forms also significantly affect the photosynthetic rate byregulating the photo-energy cost, N translocation, and enzymeactivity (Kronzucker et al., 1999). Our results revealed that thephotosynthetic parameters were affected by both chilling and Nforms, but the impact was more intense in the former (Table 3),especially in the ammonium and nitrate treatments. With ammo-nium and nitrate nutrition, this negative effect of cold stress on theplant growth and carbon assimilation was attributed to an inevi-table limitation of CO2 diffusion in leaf due to the closure of sto-mata. Especially, the reason for a higher Pn under cold stress in theglycine supplied rice plants may be as follows: (1) compared to theplants provided with the ammonium and nitrate nutrition, theglycine supplied rice maintained a higher gs and had less reductionof Ci under the cold stress, (2) the contents of leaf Chl-a, Chl-b, andcarotenoid in the glycine treated rice were significant higher thanin the plants treated with ammonium and nitrate. Both the factorswere beneficial for the enhanced photosynthesis and for the for-mation of high rice biomass.

Chilling injury, such as that manifested as the inhibition ofagronomic and physiological characters, is a physiologicaldysfunction observed in plants when they are exposed to lowtemperatures (Allen and Ort, 2001). Sonoike (1996) attributed it tothe selective photo-inhibition of either PSII or PSI, which wasclosely related to leaf Chl fluorescence parameters before thevisible injury could be observed. Our results demonstrated thatglycine maintained a high rate of photosynthesis under cold stress.However, the underlying reason for this observation remainsintricate. Mauro et al. (1997) reported that cold stress significantlyaffects the reaction center and the electron transfer activity of thePSII antenna, which causes the photo-inhibition and photo-damageand, thereby, reduces the capture capability of the reaction centerfor the excitation energy. In our study, the chlorophyll fluorescenceparameters (for example, Fv/Fm, FPSII, qP, and qN) measured afterthe imposition of chilling stress revealed that the induction ofsignificant changes before the visible injury. Especially, for theammonium and nitrate treatments, the proportions of energydistributed to P and Ex were significantly decreased, but that allo-cated to the antenna system (D) increased under the cold stress.The extra energy caused the partial inactivation of the photo-chemical reaction as well as the damage of antenna system, whichwas not beneficial for the conversion of the energy captured by leafto chemical energy and increased the thermal dissipation. Incontrast, glycine greatly increased the values of FPSII and qP in therice seedlings. It indicated that glycine could increase the propor-tion of open PSII reaction centers, and maximized the light captureefficiency. Some authors have demonstrated that the enhancedphotochemical efficiency significantly strengthened the photo-synthetic electron transport efficiency and alleviated the photo-inhibition induced by cold stress (Martínez-Carrasco et al., 2002).In our study, the glycine-treated plants maintained the highphotochemical efficiency by increasing the energy distributed tothe photochemical reaction (P) and reducing the proportiondistributed to the antenna system (D). Non-photochemicalquenching (qN), which has often been used as a self-protectivemechanism by the chlorophyll and thermal dissipation canactively suppress the photo-inhibition under cold stress. Therefore,

the development of high a qN process in GN-L treatment wasbeneficial to the release of extra energy, thus, strengthening theresistance of the thylakoid membranes to cold injury. Thestrengthened resistance further preserved the integrity of chloro-plasts and the maintenance of higher content of the pigment, ul-timately resulting in a higher Pn.

5. Conclusions

In conclusion, our study demonstrated that the strengthenedtolerance of glycine-treated rice to cold stress was closely associ-ated with its higher dry matter accumulation and photosynthesis,which was a result of an increase in the root N uptake ability,contents of osmoprotectants, antioxidant defense system, andphotochemical efficiency. With global climate changes, rice will bemore frequently exposed to extremely low temperatures; there-fore, improving the cold tolerance of rice by optimizing the form inwhich N is applied, especially the organic N, should deserved moreattention in future studies. The results of the present study alsoprovide new ideas for improving the plant tolerance to extremetemperatures by nutrient resource management in the coldregions.

Author Contribution Statement

Cao XC and Jin QY conceived and designed research. Cao XC,Zhong C and Zhu LF conducted experiments. Wu LH contributedanalytical tools. Cao XC, Zhong C, and Zhang JH analyzed data. CaoXC, Zhong C and Sajid Hussain wrote the manuscript. All authorsread and approved the manuscript.

Acknowledgments

This work was funded by the Natural Science Foundation ofZhejiang Province (No. LQ15C130004), National Basic ResearchProgram of China (No. 2015CB150502), National Natural ScienceFoundation of China (No. 31172032, 31270035).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2017.01.008

References

Agarwal, S., Pandey, V., 2004. Antioxidant enzyme responses to NaCl stress in Cassiaangustifoli. Biol. Plant. 48, 555e560.

Alam, B., Jacob, J., 2002. Overproduction of photosynthetic electrons is associatedwith chilling injury in green leaves. Photosynthetica 40, 91e95.

Allen, D.J., Ort, D.R., 2001. Impacts of chilling temperatures on photosynthesis inwarm-climate plants. Trends Plant Sci. 6, 36e42.

Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, andsignal transduction. Annu. Rev. Plant Biol. 55, 373e399.

Ashraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improvingplant abiotic stress resistance. Environ. Exp. Bot. 59, 206e216.

Benavides, M.P., Marconi, P.L., Gallego, S.M., Comba, M.E., Tomaro, M.L., 2000.Relationship between antioxidant defence systems and salt tolerance in Sola-num tuberosum. Aust. J. Plant Physiol. 27, 273e278.

Britto, D.T., Kronzucker, H.J., 2002. NH4þ toxicity in higher plants: a critical review.

J. Plant Physiol. 159, 567e584.Cao, X.C., Wu, L.H., Yuan, L., Li, X.Y., Zhu, Y.H., Jin, Q.Y., 2015. Uptake and uptake

kineticsof nitrate, ammonium and glycine by pakchoi (Brassica Campestris L.ssp. Chinensis L. Makino) seedlings. Sci. Hortic. 186, 247e253.

Demmig-Adams, B., Adams, W.W., Barker, D.H., Logan, B.A., Bowling, D.R.,Verhoeven, A.S., 1996. Using chlorophyll fluorescence to assess the fraction ofabsorbed light allocated to thermal dissipation of excess excitation. Physiol.Plant 98, 253e264.

Erdal, S., 2012. Androsterone-induced molecular and physiological changes inmaize seedlings in response to chilling stress. Plant Physiol. Biochem. 57, 1e7.

Faize, M., Burgos, L., Faize, L., Piqueras, A., Nicolas, E., Barba-Espin, G., Clemente-Moreno, M.J., Alcobendas, R., Artlip, T., Hernandez, J.A., 2011. Involvement of

C. Xiaochuang et al. / Plant Physiology and Biochemistry 112 (2017) 251e260260

cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improvedtolerance against drought stress. J. Exp. Bot. 62, 2599e2613.

Feng, H., Yan, M., Fan, X., Li, B., Shen, Q., Miller, A.J., Xu, G., 2011. Spatial expressionand regulation of rice high affinity nitrate transporters by nitrogen and carbonstatus. J. Exp. Bot. 62, 2319e2332.

Genisel, M., Turk, H., Erdal, S., 2013. Exogenous progesterone application protectschickpea seedlings against chilling-induced oxidative stress. Acta Physiol. Plant35, 241e251.

Guo, S., Brück, H., Sattelmacher, B., 2002. Effects of supplied nitrogen form ongrowth and water uptake of French bean (Phaseolus vulgaris L.) plants. PlantSoil 239, 267e275.

Guo, S., Zhou, Y., Shen, Q., Zhang, F., 2007. Effect of ammonium and nitrate nutritionon some physiological processes of higher plants. Plant Biol. 9, 21e29.

Huang, M., Guo, Z., 2005. Responses of antioxidative system to chilling stress in tworice cultivars differing in sensitivity. Biol. Plant 49, 81e84.

Kaiser, W.M., Huber, S.C., 2001. Post-translational regulation of nitrate reductase:mechanism, physiological relevance and environmental triggers. J. Exp. Bot. 52,1981e1989.

Kronzucker, H.J., Siddiqi, M.Y., Glass, A.D.M., Kirk, G.J.D., 1999. Nitrate-ammoniumsynergism in rice. A subcellular flux analysis. Plant Physiol. 119, 1041e1045.

Kuk, Y., Shin, J.S., Whang, T.E., Guh, J.O., 2002. Mechanisms of chilling tolerance inrelation to antioxidative enzymes in rice. Korean J. Crop Sci. 47, 341e351.

Lambers, H.L., Chapin, F.S., Pons, T.L., 2008. Plant Physiological Ecology. Springer,New York, USA, pp. 1e9.

Lawlor, D.W., 2002. Carbon and nitrogen assimilation in relation to yield: mecha-nisms are the key to understanding production systems. J. Exp. Bot. 53,773e787.

Li, Y., Ren, B., Yang, X., Xu, G., Shen, Q., Guo, S., 2012. Chloroplast downsizing undernitrate nutrition restrained mesophyll conductance and photosynthesis in rice(Oryza sativa L.) under drought conditions. Plant Cell Physiol 53, 892e900.

Liu, Z., Li, Y., Hou, H., Zhu, X., Rai, V., He, X., Tian, C., 2013. Differences in thearbuscular mycorrhizal fungi-improved rice resistance to low temperature attwo N levels: aspects of N and C metabolism on the plant side. Plant Physiol.Biochem 71, 87e95.

Martínez-Carrasco, R., S�anchez-Rodriguez, J., P�erez, P., 2002. Changes in chlorophyllfl uorescence during the course of photoperiod andin response to drought inCasuarina equiseti folia Forst. And Forst. Photosynthetica 40, 363e368.

Mauro, S., Dainsese, P., Lannoye, R., Bassi, R., 1997. Cold-resistant and cold-susceptible maize lines differ in the phosphorylation of the photo - systemⅡsubunit, CP29. Plant Physiol. 115, 171e180.

Nagasuga, K., Murai-Hatano, M., Kuwagata, T., 2011. Effects of low root temperatureon dry matter production and root water uptake in rice plants. Plant Prod. Sci.14, 22e29.

N€asholm, T., Kielland, K., Ganeteg, U., 2009. Uptake of organic nitrogen by plants.New Phytol. 182, 31e48.

Nayyar, H., Chander, K., Kumar, S., Bains, T., 2005. Glycine betaine mitigates coldstress damage in Chickpea. Agron. Sustain. Dev. 25, 381e388.

Oliver, S.N., Van Dongen, J.T., Alfred, S.C., Mamun, E.A., Zhao, X., Saini, H.,Fernandes, S.F., Blanchard, C.L., Sutton, B.G., Geigenberger, P., Dennis, E.S.,

Dolferus, R., 2005. Cold-induced repression of the rice anther-specific cell wallinvertase gene OSINV4 is correlated with sucrose accumulation and pollensterility. Plant Cell Environ. 28, 1534e1551.

Ploschuk, E.L., Bado, L.A., Salinas, M., Wassner, D.F., Windauer, L.B., Insausti, P., 2014.Photosynthesis and fluorescence responses of Jatropha curcas to chilling andfreezing stress during early vegetative stages. Environ. Experi. Bot. 102, 18e26.

Sachadyn-Kr�ol, M., Materska, M., Chilczuk, B., Kara, M., Jakubczyk, A., Perucka, I.,Jackowska, I., 2016. Ozone-induced changes in the content of bioactive com-pounds and enzyme activity during storage of pepper fruits. Food Chem. 211,59e67.

Shao, H., Chu, L., Shao, M., Jaleel, C., Mi, H., 2008. Higher plant antioxidants andredox signaling under environmental stresses. CR. Biol. 331, 433e441.

Shimono, H., Hasegawa, T., Iwama, K., 2002. Response of growth and grain yield inpaddy rice to cool water at different growth stages. Field Crops Res. 73, 67e79.

Shimono, H., Hasegawa, T., Fujimura, S., Iwama, K., 2004. Responses of leaf photo-synthesis and plant water status to low water temperature at different growthstages. Field Crops Res. 89, 71e83.

Sims, L., Pastor, J., Lee, T., Dewey, B., 2012. Nitrogen, phosphorus and light effects ongrowth and allocation of biomass and nutrients in wild rice. Oecologia 170,65e76.

Sonoike, K., 1996. Photoinhibition of photosystem I: its physiological significance inthe chilling sensitivity of plants. Plant Cell Physiol. 37, 239e247.

Suzuki, K., Nagasuga, K., Okada, M., 2008. The Chilling injury induced by high roottemperature in the leaves of rice seedlings. Plant Cell Physiol. 49, 433e442.

Tan, D.X., Hardeland, R., Manchester, L.C., 2012. Functional roles of melatonin inplants, and perspectives in nutritional and agricultural science. J. Exp. Bot. 63,577e597.

Triantaphylides, C., Havaux, M., 2009. Singlet oxygen in plants: production,detoxification and signaling. Trends Plant Sci. 14, 219e228.

Turk, H., Erdal, S., Genisel, M., Atici, O., Demir, Y., Yanmis, D., 2014. The regulatoryeffect of melatonin on physiological, biochemical and molecular parameters incold-stressed wheat seedlings. Plant Growth Regul. 74, 139e152.

Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidantsystems in acid rain-treated bean plants Protective role of exogenous poly-amines. Plant Sci. 151, 59e66.

Wang, H.J., Wu, L.H., Tao, Q.N., Miller, D.D., Welch, R.M., 2009. Glutamine nitrogenand ammonium nitrogen supplied as a nitrogen source is not converted intonitrate nitrogen of plant tissues of hydroponically grown Pak-Choi (Brassicachinensis L.). J. Food Sci. 74, 21e23.

Xu, W., Cui, K., Xu, A., Nie, L., Huang, J., Peng, S., 2015. Drought stress conditionincreases root to shoot ratio via alteration of carbohydrate partitioning andenzymatic activity in rice seedlings. Acta Physiol. Plant 37, 9.

Yamamoto, A., Shim, I., Fujihara, S., 2012. Chilling-stress responses by rice seedlingsgrown with different ammonium concentrations and its relationship to leafspermidine content. J. Plant Biol. 55, 191e197.

Yamori, W., Masumoto, C., Fukayama, H., Makino, A., 2012. Rubisco activase is a keyregulator of non-steady-state photosynthesis at any leaf temperature and, to alesser extent, of steady-state photosynthesis at high temperature. Plant J. 71,871e880.