lable at ScienceDirect

Plant Physiology and Biochemistry 75 (2014) 138e144

Contents lists avai

Plant Physiology and Biochemistry

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

Research article

Manipulation of monoubiquitin improves chilling tolerancein transgenic tobacco (Nicotiana tabacum)

Yanan Feng a,1, Meng Zhang a,1, Qifang Guo b, Guokun Wang a, Jiangfeng Gong a, Ying Xu a,Wei Wang a,*

a State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Tai’an, Shandong 271018, PR Chinab State Key Laboratory of Crop Biology, College of Agriculture, Shandong Agricultural University, Tai’an, Shandong 271018, PR China

a r t i c l e i n f o

Article history:Received 12 September 2013Accepted 7 November 2013Available online 19 November 2013

Keywords:Chilling tolerancePhotosynthesisReactive oxygen speciesTransgenic tobacco plantsUbiquitinWheat

Abbreviations: APX, ascorbate peroxidase; CAT, catenzyme; E2, ubiquitin-conjugating enzyme; E3, ubimaximal efficiency of PSII photochemistry; MDA, maltetrazolium; Pn, photosynthetic rate; POD, peroxidasPSII; ROS, reactive oxygen species; SOD, superoxitransgenic plants carrying the recombinant construccontrol, carrying the recombinant construct of theunder the control of CaMV 35S promoter and the nopsequences in the sense orientation; Ub, ubiquitin; UPSWT, wild-type.* Corresponding author. Tel.: þ86 538 8246166; fax

E-mail addresses: wangw@sdau.edu.cn, wangweis1 Yanan Feng and Meng Zhang contributed equally

0981-9428/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2013.11.003

a b s t r a c t

Ubiquitin (Ub) is amultifunctional protein thatmainly functions to tag proteins for selective degradationbythe 26Sproteasome.Wecloned anUbgene TaUb2 fromwheat (Triticumaestivum L.) previously. To study thefunction of TaUB2 in chilling stress, sense andantisenseUb transgenic tobaccoplants (Nicotiana tabacum L.),as well as wild type (WT) and vector control b-glucuronidase (T-GUS) plants, were used. Under stress, leafwilting in sense plants was significantly less than in controls, but more severe in antisense plants.Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) insense plants were greater than controls, but lower in antisense plants during chilling stress and recovery.Lesswilting in sense plants resulted from improvedwater status, whichmaybe related to the accumulationof proline and solute sugar. Furthermore, as indicated by electrolyte leakage, membrane damage understress was less in sense plants and more severe in antisense plants than controls. Consistent with elec-trolyte leakage, the malondialdehyde (MDA) content was less in sense plants, but more in antisense plantscompared to controls. Meanwhile, the less accumulation of reactive oxygen species (ROS) and the greaterantioxidant enzyme activity in sense plants implied the improved antioxidant competence by the over-expression of monoubiquitin gene Ta-Ub2 from wheat. We suggest that overexpressing Ub is a usefulstrategy to promote chilling tolerance. The improvement of ROS scavenging may be an important mech-anism underlying the role of Ub in promoting plants tolerant to chilling stress.

� 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction

Chilling stress is an adverse environmental factor that limits thegeographical distribution and productivity of chilling sensitive spe-cies. Plants have evolved a number ofmechanisms to response to thechanging environment rapidly and developed a variety of defensestrategies to protect themselves from chilling stress. A large number

alase; E1, ubiquitin-activatingquitin-protein ligase; Fv/Fm,ondialdehyde; NBT, nitrobluee; VPSII, actual efficiency ofde dismutase; pBI-GUS, thet of GUS gene; T-GUS, vectorb-glucuronidase gene alonealine synthase 30 termination, Ub/26S proteasome system;

: þ86 538 8242288.dau@yahoo.com (W. Wang).to this work.

son SAS. All rights reserved.

of stress-inducible genes have been identified by the combination ofmolecular and genomic methods (Cushman and Bohnert, 2000; Vijand Tyagi, 2007). However, the functional mechanisms of thesegenes with regards to either stress tolerance or sensitivity in higherplants are largely unknown. Therefore, it is critical to uncover theroles of stress-related genes to develop transgenic plants that haveimproved tolerance to unfavorable growing conditions.

Ubiquitination is the posttranslational attachment of ubiquitin(Ub), a 76-amino acid protein, to a wide range of target proteins(Pickart and Eddins, 2004). The best known ubiquitination pathwayin higher plants is the Ub/26S proteasome system (UPS), whichleads to the rapid degradation of substrate proteins (Moon et al.,2004; Dreher and Callis, 2007). In the UPS, Ub is attached to sub-strate proteins in three consecutive steps: (1) Ub is activated by anUb-activating enzyme (E1); (2) activated Ub is then transferred toan Ub-conjugating enzyme (E2); and (3) it becomes covalentlyattached to the substrate protein by an Ub ligase (E3).

The ubiquitination system appears to be present in all eukary-otic cells and is implicated in many cellular processes, including

Fig. 2. Photosynthetic performances of different transgenic plants and controls underchilling stress. Error bars represent standard deviation of five plants from each line. (a)Net photosynthetic rate (Pn, mmol CO2 m�2 s�1); (b) actual efficiency of PSII (VPSII); (c)maximal efficiency of PSII photochemistry (Fv/Fm). 0 d, before chilling stress; 1 d,chilling stress for 1 day; 3 d, chilling stress for 3 days; 5 d, chilling stress for 5days; R1 d, recovery for 1 day; R 3 d, recovery for 3 days. Each histogram represents amean � standard error of three independent experiments (n ¼ 3). Different lettersindicate significant differences between treatments (P < 0.05).

Y. Feng et al. / Plant Physiology and Biochemistry 75 (2014) 138e144 139

differentiation, cell division, hormonal responses, and biotic andabiotic stress responses. Ub is induced by various stresses in plants;however, there have been very few studies thus far on the effects ofaltered Ub gene expression on plant tolerance to chilling stress(O’Mahony and Oliver, 1999; Guo et al., 2008).

In our previous work (Guo et al., 2008; Zhang et al., 2012), amonoubiquitin gene Ta-Ub2 (AY297059) from wheat was clonedand the sense transgenic tobacco plants were generated. Theexpression of Ta-Ub2 in wheat leaves responded to drought stress.Ub-overexpressing tobacco seedlings grew more vigorously thancontrol and WT samples, and the CO2 assimilation of transgenicplants was significantly higher than that of the WT under droughtstress (Guo et al., 2008). Then, an endogenous ubiquitin gene Nt-Ub1 (DQ830978) from tobacco plants were cloned and antisenseNt-Ub1 transgenic tobacco plants with lower Ub level than WTwere generated. Overexpressing Ub enhanced the salt tolerance ofthe transgenic tobacco plants, but the opposite effect was observedin plants with repressed Ub expression (Zhang et al., 2012). In thisstudy, the different responses among sense, antisense and wildtype to chilling stress were investigated. The represented resultssuggested that Ubwas involved in the tolerance of plants to chillingstress. The ROS scavenging may be one of the most importantmechanisms underlying the alleviated damage of chilling stress totobacco plants by overexpressing Ub.

2. Results

2.1. Effects of chilling stress on growth performance of thetransgenic tobacco plants and controls

Four-month-old tobacco plants, including sense transgenic to-bacco plants (T-2 and T-11), antisense transgenic tobacco plants(AT-1 and AT-3) and wild type (WT) were exposed to 4 �C tem-perature stress. Under normal 25 �C conditions, no difference be-tween the transgenic and WT plants was observed (Fig. 1a). Afterbeing subjected to 4 �C temperature for 1 day, all plants wereseverely wilted. The wilting was more severe in antisense Nt-Ub1transgenic lines, and less in sense Ta-Ub2 transgenic lines whencompared to WT plants (Fig. 1b). A similar but more prominentdifference was observed when the plants were subjected to 4 �Ctemperature for 3 days (Fig. 1c). Interestingly, the wilting symptomalmost disappeared in all plants when subjected to 4 �C tempera-tures for 5 days (Fig. 1d). This may result from the cold acclimationor the adaptation of the plants to chilling stress.

2.2. Effects of chilling stress on photosynthetic gas exchange andPSII photochemistry in different transgenic plants and controls

At 25 �C, no significant difference was observed in leaf photo-synthetic rate (Pn) among all tobacco varieties (Fig. 2a). During 10 �C

Fig. 1. The morphological change of the transgenic tobacco plants and wild plants under normal and chilling stress temperatures (a) untreated plants at 25 �C; (b) chilling stress for1 day; (c) chilling stress for 3 days; (d) chilling stress for 5 days. In each picture, the left two plants, T-2 and T-11 were sense Ta-Ub2 transgenic tobacco lines; the right two plants, AT-1 and AT-3 were antisense Nt-Ub1 transgenic tobacco lines and the middle plant was wild types (WT).

Fig. 3. Effects of chilling stress on proline content (a) and soluble sugar content intransgenic and control plants (4 �C). 0 d, before chilling stress; 1 d, chilling stress for 1day; 3 d, chilling stress for 3 days; 5 d, chilling stress for 5days; R 1 d, recovery for 1day; R 3 d, recovery for 3 days. Each histogram represents a mean � standard error ofthree independent experiments (n ¼ 3). Different letters indicate significant differ-ences between treatments (P < 0.05).

Y. Feng et al. / Plant Physiology and Biochemistry 75 (2014) 138e144140

chilling stress, Pn decreased gradually and this decreasewas greaterin antisense plants, but less in sense plants than that in WT andT-GUS controls (Fig. 2a). The most significant difference betweenthemwas observed at 5 d after chilling stress treatment. After beingallowed to recover at 25 �C for 3 days, the Pn of all tobacco plantsreturned to a normal level, with faster recovery in sense plants butslower in antisense tobacco plants than that in controls. Similarresultswere observed in the responses of the actual efficiency of PSII(VPSII) (Fig. 2b). However, the maximal efficiency of PSII photo-chemistry (Fv/Fm) was not sensitive to 10 �C low temperature, andno significant difference was observed between different tobaccovarieties (Fig. 2c), suggesting no permanent damage was made inPSII by chilling stress in this experiment.

2.3. Effects of chilling stress on the accumulations of proline andsoluble sugar in transgenic plants and controls

It has been demonstrated that the accumulation of proline andsoluble sugar is involved in the improved tolerance of plants to saltand cold stresses (Shan et al., 2007; Huang et al., 2009). Beforechilling stress, the proline and soluble sugar contents were similarbetween transgenic lines and controls (Fig. 3a). During the chillingstress treatments, the proline content increased gradually in alltobacco plants, reaching its highest content at 5 d after chillingstress, and then returned to original levels during the 3 daysrecovery. Compared with the two controls, the proline increasewasgreater in sense tobacco plants but less in antisense tobacco plants.Similar changes were observed in soluble sugar content (Fig. 3b).

2.4. The different responses of antioxidative ability and membranestability between transgenic tobacco plants and controls to chillingstress

Seven-week-old intact seedlings of transgenic lines and controllines grown onMSmediumwere used to detect superoxide radicalsO2$

� and H2O2 accumulations. We determined O2$� by nitroblue

tetrazolium (NBT) staining and the accumulation of H2O2 by3,3-diaminobenzidine (DAB) staining. As shown in Fig. 4aed,chilling stress increased the accumulations of both O2$

� and H2O2.Compared with controls, lighter brown and blue precipitates weredetected in the sense plants, but deeper color were observed inantisense tobacco plants under both stress and normal conditions.The O2$

� production rate (Fig. 4e) and Quantifications of H2O2accumulation (Fig. 4f) showed the same results as the stainingmethod. These data revealed that overexpression of Ta-Ub2reduced the accumulation of reactive oxygen species (ROS) in sensetransgenic tobacco plants. However, after recovery, the level of ROSwas almost returned to the original level, suggesting no permanentdamage was made in tobacco plants by this stress condition as thatin PSII (Fig. 2b and c).

Chilling stress injures cell membranes, which results in elec-trolyte leakage from the cells. During the chilling stress treatments,the level of electrolyte leakage (%) gradually increased to its highestlevel at the 5 d and decreased during recovery (Fig. 5a). Comparedwith WT and T-GUS, the electrolyte leakage level was lower insense tobacco plants but higher in antisense tobacco plants. Toassess the lipid peroxidation, we measured the malondiadehyde(MDA) level in tobacco leaves and the results were similar to thechanges of electrolyte leakage (Fig. 5b).

2.5. Effects of chilling stress on the activities of antioxidant enzymesin transgenic plants and controls

Four antioxidant enzymes, including APX, CAT, superoxide dis-mutase (SOD) and POD, were examined (Fig. 6). APX activity

increased to the maximum levels after 1 day of the chilling stresstreatment, decreased to its lowest levels at the 5 d, and began torecover at the first day after the recovery, and decreased again tothe original levels (Fig. 6a). Similar changes were observed in CATand POD activities (Fig. 6b and d); however, the SOD activity alwaysincreased during chilling stress in all plants and decreased slightlyduring recovery (Fig. 6c). Compared toWTand T-GUS, the activitiesof all antioxidant enzymes were higher in sense lines, but lower inantisense lines. For instance, when treated with 4 �C for 5 days, SODactivity of sense seedlings was 16.19%e25.72% higher than controlsunder chilling stress (Fig. 6c).

3. Discussion

3.1. Overexpression of Ta-Ub2 gene enhances the chilling toleranceof transgenic tobacco plants

Plant growth and productivity are greatly affected by abioticstresses such as drought, salinity, and low temperature, etc. Thesestresses have deleterious effects on protein structure and functionin plant cells by accelerating protein damage (Bartoli et al., 2004). Ingeneral, themajority of these damaged proteins are tagged by Ub toproduce Ub-conjugates and then degraded by the Ub/26S protea-some (Pickart, 2001; Weissman, 2001; Poppek and Grune, 2006).Ub is induced by various stresses in plants (O’Mahony and Oliver,1999; Genschik et al., 1992; Sun and Callis, 1997), which wasconsidered to be necessary to tag the damaged proteins (O’Mahonyand Oliver, 1999; Garbarino et al., 1995).

To study the functions of Ub in stress tolerance of the plants, wecloned an Ub gene Ta-Ub2 from wheat and produced sense trans-genic tobacco plants with overexpression of Ub (Guo et al., 2008).Meanwhile, to obtain the transgenic tobacco plants with inhibitionof Ub expression, another Ub gene Nt-Ub1was cloned from tobaccoplants and antisense transgenic tobacco lines were generated(Zhang et al., 2012). As shown in Fig. 1, chilling stress resulted insevere wilting in all tobacco plants examined. Compared withcontrols, the sense plants displayed a better water status; reversely,

Fig. 4. ROS accumulation in different transgenic plants under chilling stress (4 �C). The superoxide radical (O2$�) (a and c) and H2O2 (b and d) content were used to show the ROS

accumulation of plants under normal condition (a and b) and after one-day chilling stress (c and d). (e) H2O2 content; (f) O2$� production rate. 0 d, before chilling stress; 1 d, chilling

stress for 1 day; 3 d, chilling stress for 3 days; 5 d, chilling stress for 5days; R1d, recovery for 1 day; R3d, recovery for 3 days. Each histogram represents a mean � standard error ofthree independent experiments (n ¼ 3). Different letters indicate significant differences between treatments (P < 0.05).

Y. Feng et al. / Plant Physiology and Biochemistry 75 (2014) 138e144 141

the antisense Nt-Ub1 transgenic plants showed worse wilting.Meanwhile, the Pn and VPSII in Fig. 2 also indicated the betterphysiological status in sense plants than controls and antisenseplants. The improved water status in sense plants may be related tothe accumulation of proline and soluble sugars (Fig. 3), becauseboth of them are the important compatible solutes involved inosmotic adjustment (Sánchez et al., 1998). These results suggestedthe involvement of Ub and/or the UPS in plant chilling tolerance.Integrating the results in this paper with the previous data (Guoet al., 2008; Zhang et al., 2012), we suggest that the over-expression of Ub is a beneficial strategy to improve stress tolerancein plants.

3.2. ROS scavenging may be one of the important mechanismsunderlying the improvement of chilling tolerance in plants

Extreme environments often adversely affect plant growth byincreasing free radicals that encourage damage of the plant cells.Removing these proteins by various quality control pathways is

critical for cell survival (Kopito, 2000; Kostova and Wolf, 2003).Evidences from previous studies indicated that stress damage isoften mediated by reactive oxygen species (ROS) in plant systems(Scandalios, 1993), and our results in Fig. 4 also indicated theincreased ROS (including O2$

� and H2O2) accumulation duringchilling stress treatments.

Under oxidative stress conditions, ROS causes damage to pro-teins, lipids, carbohydrates and DNA, and ultimately results in celldeath (Mittler et al., 2004; Foyer and Noctor, 2005). MDA is aproduct of lipid peroxidation, which can lead to electrolyte leakagefrom cells. Chilling stress resulted in MDA (Fig. 5b) accumulations,concomitant with a resultant increase of electrolyte leakage(Fig. 5a), a major malicious effect of chilling stress on plant cells(Steponkus, 1984). The results of electrolyte leakage and MDAcontents indicated the improved antioxidative ability and mem-brane stability by overexpression of Ub.

To maintain ROS at low steady-state levels, plants have evolvedantioxidative systems to keep ROS under control (Scandalios, 1993;Asada, 1992). Several main antioxidant enzymes, including SOD,

Fig. 5. Effects of chilling stress on the electrolyte leakage, MDA contents in transgenicplants and controls. (a) Electrolyte leakage; (b) MDA content. 1e3: represent T-2, T-11,WT, under normal condition; 4e6: represent T-2, T-11, WT, under chilling stress; 7e9:represent T-2, T-11, WT, under recover normal condition. 0 d, before chilling stress; 1 d,chilling stress for 1 day; 3 d, chilling stress for 3 days; 5 d, chilling stress for 5days; R1d,recovery for 1 day; R3d, recovery for 3 days. Each histogram represents amean � standard error of three independent experiments (n ¼ 3). Different lettersindicate significant differences between treatments (P < 0.05).

Y. Feng et al. / Plant Physiology and Biochemistry 75 (2014) 138e144142

CAT, POD and APX, participate in this process. As shown in Fig. 6,chilling stress upregulated the activity of all these antioxidant en-zymes, but when compared to that in controls and antisense Nt-Ub1 lines, their activities were always higher in sense plants. Thismay be related to the less ROS accumulation in these plants (Fig. 4),and suggest the function of Ub in the improvement of plant anti-oxidative systems.

In conclusion, our research suggested that Ub was involved inthe tolerance of plants to chilling stress, and overexpression ofTa-Ub2was an effective strategy to improve plant chilling tolerance.The changed antioxidative ability may be one of the most impor-tant mechanisms underlying the functions of Ub in the plantchilling tolerance.

Fig. 6. Activities of APX (a), CAT (b), SOD (c) and POD (d) in different transgenic plantsexposed to chilling stress (4 �C). 0 d, before chilling stress; 1 d, chilling stress for 1 day;3 d, chilling stress for 3 days; 5 d, chilling stress for 5 days; R1d, recovery for 1 day;R3d, recovery for 3 days. Each histogram represents a mean � standard error of threeindependent experiments (n ¼ 3). Different letters indicate significant differencesbetween treatments (P < 0.05).

4. Materials and methods

4.1. Plant materials and growth conditions

The sense Ta-Ub2 transgenic tobacco (Nicotiana tabacum) linesT-2, T-11 and T-13, antisense Nt-Ub1 transgenic tobacco lines AT-1,AT-3 and AT-10, wide type (WT) and T-GUS (vector control, carryingthe recombinant construct of the b-glucuronidase (GUS) genealone) were used. Both the sense and the antisense transgenicplants were T2 or T3 homozygous generation, and the expressedheterogenous gene was identified by polymerase chain reaction(PCR) and immunological blot (Zhang et al., 2012).

For full-growntobaccoplants, seedswere sown inpots (8�10cm)containing vermiculite soaked with half-strength Hoagland nutrientsolution (the irrigation was enough to avoid water stress and main-tained the natural growth) and grown at 25/20 �C (day/night) with a14 h photoperiod (300e400 mmol photonsm�2 s�1) in a greenhouse.

Tobacco infancy seedlings were used to detected ROS accumu-lation. Seeds were treated with 70% ethanol for 5 min and sterilizedwith 4% NaClO for 10 min. After washing six times with sterilewater, the seeds were planted on solidified MurashigeeSkoog (MS)medium. Different genotypes were grown on the same plate during

chilling stress till used. These infancy seedlings were prepared forstaining detection of superoxide radical (O2$

�) and H2O2 levels.Chilling stress treatment was performed on four-month-old

plants with 7e8 leaves and infancy seedlings. The whole plantswere exposed to low temperature (4 �C or 10 �C) for 1, 3 or 5 days.After chilling stress treatment, the plants were allowed to recoverat 25 �C for 1 or 3 days.

4.2. Measurements of photosynthetic gas exchange and chlorophyllfluorescence parameters

Net photosynthetic rate (Pn) was measured with a portablephotosynthetic system (CIRAS-2, PP Systems, Herts, UK) under thecondition of the ambient CO2 concentration of 360 mL L�1, photo-synthetic photon flux density (PPFD) of 800 mmol m�2 s�1 andrelative humidity of 80%. Before Pn measurement, plants were kept

Y. Feng et al. / Plant Physiology and Biochemistry 75 (2014) 138e144 143

at 25 �C, 100 mmol photons m�2 s�1 for 30 min to induce stomata toopen, and then illuminated at PPFD of 800 mmol m�2 s�1 for 15 minto be acclimated.

The intrinsic efficiency (Fv/Fm) and actual efficiency (FPSII) ofphotosystem II (PSII) of the same tobacco leaves were measuredwith a portable fluorometer (FMS2, Hansatech, Norfolk, UK)according to the protocol described by Kooten and Snel (1990).Plants were adapted in darkness for 30 min before Fv/Fm mea-surement. The minimal fluorescence (Fo) with all PSII reactioncenters free was determined by modulated light, which was lowenough not to induce any significant variable fluorescence (Fv). Themaximal fluorescence (Fm) with all reaction centers charged wasdetermined by 0.8 s saturating light of 7000 mmol photons m�2 s�1

on a dark-adapted (adapted 30 min in darkness) leaf. Maximalphotochemical efficiency of PSII (Fv/Fm) was expressed as: Fv/Fm ¼ (Fm � Fo)/Fm.

4.3. Measurements of proline and soluble sugar contents

Proline was measured according to Shan et al. (2007).To measure the soluble sugar content, frozen leaf material

(0.3 g) was extracted with 10 mL H2O at 100 �C for 10 min. Theextracts were filtered and analyzed for soluble sugar content usingthe anthrone-sulfuric acid method. Briefly, 1 mL of the extract wasmixed with 1 mL of H2O, 0.5 mL of anthrone reagent (1 g anthroneand 50 mL ethylacetate) and 5 mL oil of vitriol, and then heated at100 �C for 1 min. After cooling, the mix was analyzed by using UVspectrophotometry at 630 nm.

4.4. Measurements of relative electric conductivity and lipidperoxidation

To measure relative electric conductivity (Cao et al., 2007), tenleaf discs (0.8 cm diameter) were put into 20mL distilled water andvacuumized for 30 min, and then surged for 3 h to measure theinitial electric conductance (S1) (25 �C). A cuvette was filled withleaf discs and distilled water, the mixture was cooked (100 �C) for30min and then reduced to room temperature (25 �C) to determinethe final electric conductance (S2). The relative electric conduc-tivity (REC) was evaluated as: REC ¼ S1 � 100/S2.

Lipid peroxidation was determined by estimating malondial-dehyde (MDA) content in 1 g leaf fresh weight (Madhava Rao andSresty, 2000).

4.5. Staining detections of superoxide radical and H2O2

accumulations

H2O2 and superoxide radical (O2$�) accumulations were

detected by the DAB and NBT staining methods (Scarpeci et al.,2008; Zong et al., 2009). The infancy seedlings were infiltratedwith 5 g L�1 3,3-diaminobenzidine (DAB) at pH 3.8 for 20 h and0.5 g L�1 nitroblue tetrazolium (NBT) for 20 h in the dark to detectH2O2 and O2$

�, respectively. Then the seedlings were decolorizedby boiling in ethanol (96%) for 10min. After cooling, the leaveswereextracted at room temperature with 60% glycerol andphotographed.

4.6. Determinations of superoxide radical production rate, H2O2

content and anti-oxidative enzyme activities

The leaf sample (1 g) was homogenized in 3 mL 50 mM sodiumphosphate buffer (pH 7.8) including 1 mM EDTA and 2% (w/v) pol-yvinylpyrrolidone (PVP). The homogenate was centrifuged at12,000 � g for 20 min at 4 �C; the supernatant was used for theimmediate determination of superoxide radical production rate,

H2O2 content, protein content and enzyme activities. All assaysweredone at 4 �C. Total soluble protein content was determined ac-cording to Bradford (1976) using BSA as a standard. The O2$

� pro-duction rate was determined by monitoring nitrite formation fromhydroxylamine in the presence of O2$

� (Elstner and Heupel, 1976).The absorbance was read at 530 nm H2O2 content was measuredaccording to Gay and Gebicki (2000). Superoxide dismutase (SOD),catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD)activities were determined as described in (Türkan et al., 2005). Allspectrophotometric analyseswere conductedwith a Shimadzu (UV-2550) spectrophotometer (Shimadzu, Japan).

4.7. Statistical analysis

At least 3 times were repeated in all the experiments. Inparticularly, for measurements of photosynthetic gas exchangeparameters and relative electric conductivity, at least 6 times wererepeated. The data were presented as the mean � standard error(SE) of three independent experiments. Statistical analysis wasconducted using the procedures of data processing system (DPS;Zhejiang University, China). Significant differences were detectedusing the two-way analysis of variance. The means were comparedusing Duncan’s multiple range tests at P < 0.05.

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (31370304) and by the Opening Foundation ofState Key Laboratory of Crop Biology (2013KF01).

References

Asada, K., 1992. Ascorbate peroxidase-a hydrogen peroxide-scavenging enzyme inplants. Physiol. Plant. 85, 235e241.

Bartoli, C.G., Gómez, F., Martínez, D.E., Guiamet, J.J., 2004. Mitochondria are themain target for oxidative damage in leaves of wheat (Triticum aestivum L.).J. Exp. Bot. 55, 1663e1669.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Anal.Biochem. 72, 248e254.

Cao, W.H., Liu, J., He, X.J., Mu, R.L., Zhou, H.L., Chen, S.Y., Zhang, J.S., 2007. Modu-lation of ethylene responses affects plant salt-stress responses. Plant Physiol.143, 707e719.

Cushman, J.C., Bohnert, H.J., 2000. Genomic approaches to plant stress tolerance.Curr. Opin. Plant Biol. 3, 117e124.

Dreher, K., Callis, J., 2007. Ubiquitin, hormones and biotic stress in plants. Ann. Bot.99, 787e822.

Elstner, E.F., Heupel, A., 1976. Inhibition of nitrite formation from hydrox-ylammoniumchloride: a simple assay for superoxide dismutase. Anal. Biochem.70, 616.

Foyer, C.H., Noctor, G., 2005. Redox homeostasis and antioxidant signaling: ametabolic interface between stress perception and physiological responses.Plant Cell 17, 1866e1875.

Garbarino, J.E., Oosumi, T., Belknap, W.R., 1995. Isolation of a polyubiquitin promoterand its expression in transgenic potato plants. Plant Physiol. 109, 1371e1378.

Gay, C., Gebicki, J.M., 2000. A critical evaluation of the effect of sorbitol on the ferric-xylenol orange hydroperoxide assay. Anal. Biochem. 284, 217e220.

Genschik, P., Parmentier, Y., Durr, A., Marbach, J., Criqui, M.-C., Jamet, E., Fleck, J.,1992. Ubiquitin genes are differentially regulated in protoplast-derived culturesof Nicotiana sylvestris and in response to various stresses. Plant Mol. Biol. 20,897e910.

Guo, Q., Zhang, J., Gao, Q., Xing, S., Li, F., Wang, W., 2008. Drought tolerance throughoverexpression of monoubiquitin in transgenic tobacco. J. Plant Physiol. 165,1745e1755.

Huang, J.G., Yang,M., Liu, P., Yang, G.D.,Wu, C.A., Zheng, C.C., 2009.GhDREB1 enhancesabiotic stress tolerance, delays GA-mediated development and represses cyto-kinin signalling in transgenic Arabidopsis. Plant Cell Environ. 32, 1132e1145.

Kooten, O., Snel, J.F., 1990. The use of chlorophyll fluorescence nomenclature inplant stress physiology. Photosynth. Res. 25, 147e150.

Kopito, R.R., 2000. Aggresomes, inclusion bodies and protein aggregation. TrendsCell Biol. 10, 524e530.

Kostova, Z., Wolf, D.H., 2003. For whom the bell tolls: protein quality control of theendoplasmic reticulum and the ubiquitin-proteasome connection. EMBO J. 22,2309e2317.

Y. Feng et al. / Plant Physiology and Biochemistry 75 (2014) 138e144144

Madhava Rao, K., Sresty, T., 2000. Antioxidative parameters in the seedlings ofpigeonpea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses.Plant Sci. 157, 113e128.

Mittler, R., Vanderauwera, S., Gollery, M., Van Breusegem, F., 2004. Reactive oxygengene network of plants. Trends Plant Sci. 9, 490e498.

Moon, J., Parry, G., Estelle, M., 2004. The ubiquitin-proteasome pathway and plantdevelopment. Plant Cell 16, 3181e3195.

O’Mahony, P.J., Oliver, M.J., 1999. The involvement of ubiquitin in vegetativedesiccation tolerance. Plant Mol. Biol. 41, 657e667.

Pickart, C.M., Eddins, M.J., 2004. Ubiquitin: structures, functions, mechanisms. Bba-mol. Cell Res. 1695, 55e72.

Pickart, C.M., 2001. Mechanisms underlying ubiquitination. Ann. Rev. Bio 70, 503e533.

Poppek, D., Grune, T., 2006. Proteasomal defense of oxidative protein modifications.Antioxid. Redox Sign. 8, 173e184.

Sánchez, F.J., Manzanares, M.a., de Andres, E.F., Tenorio, J.L., Ayerbe, L., 1998. Turgormaintenance, osmotic adjustment and soluble sugar and proline accumulationin 49 pea cultivars in response to water stress. Field Crop Res. 59, 225e235.

Scandalios, J.G., 1993. Oxygen stress and superoxide dismutases. Plant Physiol. 101,7e12.

Scarpeci, T.E., Zanor, M.I., Carrillo, N., Mueller-Roeber, B., Valle, E.M., 2008. Gener-ation of superoxide anion in chloroplasts of Arabidopsis thaliana during activephotosynthesis: a focus on rapidly induced genes. Plant Mol. Biol. 66, 361e378.

Shan, D.P., Huang, J.G., Yang, Y.T., Guo, Y.H., Wu, C.A., Yang, G.D., Gao, Z., Zheng, C.C.,2007. Cotton GhDREB1 increases plant tolerance to low temperature and isnegatively regulated by gibberellic acid. New Phytol. 176, 70e81.

Steponkus, P.L., 1984. Role of the plasma membrane in freezing injury and coldacclimation. Ann. Rev. Plant Physiol. 35, 543e584.

Sun, C.W., Callis, J., 1997. Independent modulation of Arabidopsis thaliana poly-ubiquitin mRNAs in different organs and in response to environmental changes.Plant J. 11, 1017e1027.

Türkan, _I., Bor, M., Özdemir, F., Koca, H., 2005. Differential responses of lipid per-oxidation and antioxidants in the leaves of drought-tolerant P. acutifolius Grayand drought-sensitive P. vulgaris L. subjected to polyethylene glycol mediatedwater stress. Plant Sci. 168, 223e231.

Vij, S., Tyagi, A.K., 2007. Emerging trends in the functional genomics of the abioticstress response in crop plants. Plant Biotechnol. J. 5, 361e380.

Weissman, A.M., 2001. Themes and variations on ubiquitylation. Nat. Rev. Mol. CellBio 2, 169e178.

Zhang, J., Guo, Q., Feng, Y., Li, F., Gong, J., Fan, Z., Wang, W., 2012. Manipulation ofmonoubiquitin improves salt tolerance in transgenic tobacco. Plant Biol. 14,315e324.

Zong, X.J., Li, D.P., Gu, L.K., Li, D.Q., Liu, L.X., Hu, X.L., 2009. Abscisic acid andhydrogen peroxide induce a novel maize group C MAP kinase gene, ZmMPK7,which is responsible for the removal of reactive oxygen species. Planta 229,485e495.