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Environmental and Experimental Botany 74 (2011) 255– 260

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l h omepa g e: www.elsev ier .com/ locate /envexpbot

gDREBa transgenic chrysanthemum confers drought and salinity tolerance

umei Chen1, Xinli Cui1, Yu Chen, Chunsun Gu, Hengbin Miao, Haishun Gao, Fadi Chen ∗,haolei Liu, Zhiyong Guan, Weimin Fang

ollege of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

r t i c l e i n f o

rticle history:eceived 2 August 2010eceived in revised form 9 June 2011ccepted 17 June 2011

a b s t r a c t

The drought and salinity tolerances of a creeping ground-cover chrysanthemum variety ‘Yuhuaxunzhang’were compared to the performance of its two derived transgenic lines carrying a drought-responsiveelement binding (DREB) factor from chrysanthemum designated as CgDREBa. The over-expression ofCgDREBa conferred a measure of tolerance to both stresses. The transgenic lines showed a higher survival

eywords:rought tolerancealinity toleranceransgenic chrysanthemumgDREBa

rate and were better able to retain fresh weight in the presence of stress. Activities of superoxide dismu-tase and peroxidase and the proline content were all higher in the leaves of the transgenic plants after aprolonged period of stress, but they accumulated less malondialdehyde. CgDREBa appears to function asa transcription activator of genes within the oxidative and osmotic homeostasis transduction pathways,and represents a promising candidate for a biotechnological approach to improve the level of abioticstress tolerance in plants.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Abiotic stresses, in particular drought and salinity, not onlyompromise crop quality and limit yield, but also restrict theeographical range over which crop production is viable (Thakurt al., 2010). Plant species have evolved a number of physiologicalnd molecular means to cope with adverse environmental condi-ions. Substantial progress has been made in the identification ofhe components of some of the signalling pathways involved inhe stress response. An important class of relevant transcriptionactors has been termed “drought-responsive element binding”DREB) factors, which activate the expression of a number of stress-esponsive genes by specifically binding to them. The Arabidopsishaliana DREB family includes AtCBF2/DREB1C, a negative regulatorf AtCBF1/DREB1B and AtCBF3/DREB1A expression, this gene plays

central role in stress tolerance (Novillo et al., 2004). DREB1/CBFomologues have also been identified in rice (Dubouzet et al.,003), canola (Zhao et al., 2006), soybean (Chen et al., 2009) andarley (Choi et al., 2002). The constitutive expression of DREB1enes up-regulates the expression of a suite of stress-responsive

enes, thereby increasing the plant’s tolerance to cold, drought,nd salinity stresses (Gilmour et al., 2000; Oh et al., 2007). Tobaccolants expressing AtCBF3 show improved levels of drought and cold

∗ Corresponding author. Tel.: +86 25 84395231; fax: +86 25 84395266.E-mail address: chenfd@njau.edu.cn (F. Chen).

1 Sumei Chen and Xinli Cui contributed equally to this work reported here.

098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2011.06.007

tolerance (Kasuga et al., 2004), while tomato plants transformedwith AtCBF1 are characterized by an enhanced level of catalaseactivity, which is associated with a reduction in the accumulationof the powerful oxidant hydrogen peroxide (Hsieh et al., 2002).Arabidopsis over-expressing dwarf apple MbDREB1 enhanced planttolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways (Yang et al., 2011).Over-expression of GmDREB3 enhanced tolerance to cold, drought,and high salt stresses in transgenic Arabidopsis (Chen et al., 2009).Over-expression of CkDREB from Caragana korshinski in transgenictobacco plants resulted in enhanced tolerance to high salinity andosmotic stresses (Wang et al., 2011). ARAG1, an ABA-responsiveDREB gene, plays a role in seed germination and drought toleranceof rice (Zhao et al., 2010).

Chrysanthemum (Chrysanthemum grandiflorum, sym. Chrysan-themum morifolium) is a leading ornamental species, and the yieldand quality of the plant is heavily dependent on the environmentalconditions, especially, salinity and drought. Thus the improvementof its tolerance to these abiotic stresses is a priority for breeders.The expression of DmDREBa (also referred to as CgDREBa), isolatedfrom Chrysanthema grandiflorium var. ‘Hong Feng’, can be inducedby various abiotic stresses (Yang et al., 2009). Over-expression oftwo chrysanthemum DgDREB1 group genes caused delayed flow-ering or dwarfism in Arabidopsis (Tong et al., 2009). Heterologous

expression of the AtDREB1A gene in chrysanthemum increaseddrought and salt stress tolerance by enhancing proline contentand SOD activity (Hong et al., 2006) and tolerance to heat stressvia regulating photosynthesis (Hong et al., 2009). Here, we aimed

2 xperimental Botany 74 (2011) 255– 260

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Fig. 1. The expression of CgDREBa in control (WT) and transgenic (35S:TDa4 and35S:TDa5) plants, as measured by qRT-PCR. Transcript abundance was normalized

56 S. Chen et al. / Environmental and E

o improve the stress tolerance of chrysanthemum via generatinggDREBa transgenic plants, and illustrate the regulatory pathway ofhe CgDREBa transcript factor from chrysanthemum under abiotictresses. We succeeded in characterizing two transgenic ground-over chrysanthemum lines which over-express CgDREBa, and haveeen able to show that the over-expression of the transgene con-ers an enhanced level of both drought and salinity tolerance,ver-expression of CgDREBa enhanced the activities of SOD, PODnd proline content, while decreased the MDA content. To ournowledge, it is the first report to describe transgenic expres-ion of CgDREBa from chrysanthemum conferred high salt androught tolerance via regulating several abiotic related pathways

n chrysanthemum.

. Materials and methods

.1. Plant material and growing conditions

The creeping ground-cover chrysanthemum variety ‘Yuhuaxun-hang’ and its derived CgDREBa transgenic lines are maintained byhe Chrysanthemum Germplasm Resource Preserving Centre, Nan-ing Agricultural University, China. Both wild type and transgeniclants were planted in 8 cm diameter pots filled with a 1:1 mixturef peat and perlite, and the plants were continuously illuminated∼240 �mol m−2 s−1) under a regime of 23 ± 2 ◦C, 12 h photoperiodnd 70% relative humidity. Seedlings at the 8–10 leaf stage weresed for the evaluation of stress tolerance. Each abiotic stress assay

nvolved three replicates of a set of 30 plants per line.

.2. Generation of sense CgDREBa transgenic lines

A full-length CgDREBa sequence was obtained by PCR andnserted in the sense orientation into the pCAMBIA1301 BamHInd KpnI sites, along with the CaMV 35S promoter. The transgeneas introduced into ‘Yuhuaxunzhang’ via agroinfection (Cui et al.,

009). Two sense CgDREBa transformants were derived, named5S:TDa4 and 35S:TDa5.

.3. Quantitative real-time PCR of CgDREBa

Total RNA was extracted from both the wild type and theransgenic plants using the Trizol reagent (Invitrogen, USA), andhe first strand cDNA was synthesized using M-MLV reverseranscription system (Promega, USA). Expression profiles of theransgene were obtained by quantitative RT-PCR, based on SYBRreen Real-time Master Mix (TOYOBO, Japan). The CgDREBa tar-eted primer sequences were 5′-TATGTTTCCCTTTTCTCCCT and′-CATTCAAAACGATAAACTCACA. The PCR comprised an initialenaturation of 95 ◦C/60 s, followed by 40 cycles of 95 ◦C/15 s,5 ◦C/15 s and extension at 72 ◦C for 45 s followed by 60 s at 95 ◦C.enaturation curves were obtained by ramping the temperature

rom 55–95 ◦C at 0.5 ◦C s−1, taking the presence of a single meltingeak to imply that the amplicon was fully specific. The referenceene was psaA (primer sequences: 5′-CCAATAACCACGACCGCTAAnd 5′-GGCACAGTCCTCCCAAGTAA).

.4. Evaluation of drought and salinity tolerance

Plant survival rate and morphological adaptation to droughttress were evaluated by withholding water from fully watered–10 leaf plants for a period of 11 days, followed by a post-tress recovery period of 14 days with full watering. Fresh weight

etention under drought stress was evaluated from plants grownydroponically, plants were exposed to 20% PEG6000 for six daysnd allowing a ten day recovery period. Plant survival and morpho-ogical adaptation to salinity stress were evaluated by exposure to

against the expression of the constitutively expressed psaA. Error bars based onthree replicates. Different lower case letters indicate significant (P < 0.05) differencesaccording to Duncan’s multiple range test.

200 mM NaCl over five days followed by a 14 day recovery period,while fresh weight retention was evaluated from plants culturedhydroponically under a four day exposure to 200 mM NaCl and aten day recovery period. Survival and fresh weight retention rateswere calculated following Zhang et al. (2005).

2.5. Enzyme activities, MDA and proline content measurement

The fourth and fifth leaves from the apex of control and trans-genic lines were sampled 0, 3, 5, 7, 9 and 11 days during droughtstress, and 0, 1, 2, 4, 6 and 8 days during salinity stress. Superox-ide dismutase (SOD) activity and malondialdehyde (MDA) contentwere assayed following Yin et al. (2009), peroxidase (POD) activityfollowing Pan et al. (2006) and proline content following Liu et al.(2005).

2.6. Protein content

Protein content was determined according to the method ofBradford (1976) with a minor modify using BSA as a standard.

2.7. Statistical analysis

A one-way analysis of variance (ANOVA), followed by the useof Duncan’s multiple range test (P = 0.05), was employed to assesswhether treatment means differed statistically from one another.SPSS v13.0J software was used for all statistical calculations.

3. Results

3.1. Expression of CgDREBa

The quantitative real-time PCR experiment demonstrated thatthe expression of CgDREBa in 35S:TDa4 and 35S:TDa5 was signif-icantly (P < 0.05) higher than that in the non-transgenic controlplants (Fig. 1).

3.2. Drought tolerance and physiological changes in CgDREBatransgenic chrysanthemum

By the end of the drought treatment, the control plants appearedheavily wilted, with all remaining leaves withered; in contrast,both transgenic lines appeared less affected (Fig. 2A). After thewater recovery, the survival rates of the two transgenic lines were,

respectively, 43.8% and 31.3%, while that of the control was only6.3% (Fig. 2B). The fresh weight retention rates of 35S:TDa4 was18.7%, and that of 35S:TDa5 15.7%; the equivalent rate for the con-trol was only 3.6% (Fig. 2C). SOD activity in the control increased

xperimental Botany 74 (2011) 255– 260 257

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Fig. 3. Effect of drought stress on SOD activity and proline and MDA content of con-

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apidly over the duration of the drought stress, but was significantlyP < 0.05) higher in 35S:TDa5 plants; in 35S:TDa4 plants, SOD activ-ty remained at a significant (P < 0.05) low level for the first fiveays, but thereafter significantly increased more rapidly than inhe control, reaching a peak of 78.3 U/mg by the end of the droughttress period (Fig. 3A). Before the drought stress, the proline con-ent of all three lines was uniformly low. In the control plants,t increased over the first five days, remained stable for the nextour, and then increased once more; in contrast, in the transgenicines it increased slowly over the first seven days, and acceleratedntil the end of the drought stress period (Fig. 3B). A peak in theroduction of MDA occurred in all three lines after three days ofrought stress; thereafter, the MDA content decreased graduallyFig. 3C). The MDA content of both 35S:TDa4 and 35S:TDa5 wereenerally significantly (P < 0.05) lower than in the control through-ut the drought stress period, with the level in 35S:TD4 below thatn 35S:TD5 (Fig. 3C).

.3. Salt tolerance and physiological changes in CgDREBaransgenic chrysanthemum

Control plants were more sensitive to salinity stress than thegDREBa transformants (Fig. 4A). After three days of exposure, mostf the control plant leaves were necrotic, while those of both trans-enic lines showed only lesser signs of injury. By the end of salinitytress treatment, the survival rates of 35S:TDa4 and 35S:TDa5lants were, respectively, 51.1% and 33.3%, while that of the con-rol was only 11.1% (Fig. 4B). The fresh weight retention rates for5S:TDa4, 35S:TDa5 and the control were, respectively, 12.8%, 9.3%nd just 1.1% (Fig. 4C). The lines showed distinct patterns of tem-oral variation in POD activity (Fig. 5A). In the control it peaked at063.1 U/g FW min after one day, then fell to a steady state levelf 709.8U/g FW min. In 35S:TDa4 and 35S:TDa5 plants, however, aimilar peak value was reached only after three days; followed by arief decrease and then a rapidly increase to reach markedly higher

evels than in the control plants by the end of the stress period. Pro-ine content increased over the duration of the salinity stress, withts level remaining significantly (P < 0.05) lower in 35S:TD5 than in

ontrol up to the fourth day. After this, the level rose more quicklyhan in the control in both transgenic lines and kept increasing afterour days under salt stress (Fig. 5B). The MDA content of the threeines was indistinguishable before the salinity stress. During the

trol (WT) and transgenic (35S:TDa4 and 35S:TDa5) plants. A: SOD activity. B: prolinecontent. C: MDA content. * represents significant differences (p < 0.05) between thetransgenic (35S:TDa4 and 35S:TDa5) plants and WT plants at the same time.

ig. 2. Drought tolerance assay of transgenic chrysanthemum plants. A: Control (WT) and transgenic (35S:TDa4 and 35S:TDa5) plants grown under non-stressed and droughttressed conditions. Drought-watering withheld for 11 days; recovery-normal watering for 14 days. B, C: Survival and fresh weight retention rates of WT, 35S:TDa4 and5S:TDa5 plants. Error bars based on three replicates. Different lower case letters indicate significant (P < 0.05) differences according to Duncan’s multiple range test.

258 S. Chen et al. / Environmental and Experimental Botany 74 (2011) 255– 260

Fig. 4. Salinity tolerance assay of transgenic chrysanthemum plants. A: Control (WT) and transgenic (35S:TDa4 and 35S:TDa5) plants exposed to 200 mM NaCl for 0, 1, 3and 5 days. B, C: Survival and fresh weight retention rates of WT, 35S:TDa4 and 35S:TDa5 plants. Error bars based on three replicates. Different lower case letters indicates

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ignificant (P < 0.05) differences according to Duncan’s multiple range test.

tress period, it remained stable in the transgenic lines, and at aignificant (P < 0.05) lower level than the control plants. The MDAontent of the control plants rose to a peak of 88.3 �mol/g FW bywo days, and thereafter fell gradually (Fig. 5C).

. Discussion

Genes known to enhance tolerance to drought and salinity haveeen classified into four groups: (1) those encoding antioxidantnzymes, such as SOD, POD and catalase (Ashraf, 2009); (2) thoseesponsible for the regulation of osmotica, such as �1-pyrroline--carboxy-late synthase (Majee et al., 2004); (3) those encodingtress-inducible proteins such as LEA (Raynal et al., 1999); and (4)egulatory genes, such as DREB (Pellegrineschi et al., 2004). TheREB/CBF genes are considered as ‘master switches’, since DREBroteins bind to a number of downstream stress-inducible genesincluding, in A. thaliana, rd29A, kin1, cor6, cor15a, rd17, erd10, p5cs,rd1, rd22, rd29B and LEA) and regulate their expression to adapto cold, drought and salinity stress (Maruyama et al., 2004). Con-titutive expression of DREB1/CBF in transgenic A. thaliana led tohe over-expression of a number of CRT/DRE-containing genes,liciting a higher level of tolerance to salinity and/or droughttress (Kasuga et al., 1999; Kitashiba et al., 2004; Tong et al.,009; Peng et al., 2011; Yang et al., 2011). In rice, the over-xpression of OsDREB improved the level of tolerance to droughtChen et al., 2008). Stress-inducible expression of GmDREB1 con-erred salt tolerance in transgenic alfalfa (Jin et al., 2010). GmDREB2,

soybean DRE-binding transcription factor, conferred droughtnd high-salt tolerance in transgenic plants (Chen et al., 2007).ver-expression of TsCBF1 gene from a dicotyledonous halophytehellungiella halophila conferred improved drought tolerance inransgenic maize (Zhang et al., 2010). A DRE-binding protein Tdic-RF1 from Triticum dicoccoides displayed drought response (Lucast al., 2011). Over expression of DREB/CBF factors from wheat grainsn transgenic lines showed improved survival of wheat and barleynder severe drought conditions (Morran et al., 2011). Here weave shown that the over-expression of CgDREBa also improvedhe tolerance to salinity or drought stress in chrysanthemum. The

ransgenic phenotype under stressed growing conditions showedess injury, better survival, higher fresh weight retention, suggestedhat the over-expression of CgDREBa activates a series of down-tream stress-tolerant genes.

Over-expression of GmDREB3 enhanced tolerance to cold,drought, and high salt stresses in transgenic Arabidopsis. Physio-logical analyses indicated that the fresh weight and osmolytes ofGmDREB3 transgenic Arabidopsis under cold stress were higher thanthose of wild-type controls (Chen et al., 2009). Drought and salinitystress resulted in a derived water loss and retard in plant growth(Romero-Aranda et al., 2006; Verslues et al., 2006). In present study,fresh weight retention is higher in CgDREBa transgenic plants undersalinity and drought stress than that of control plants, which mightdue to less water loss or increase in proline levels.

Free proline in plants plays the roles of osmotic adjustment,protection of cellular macromolecules and scavenging of hydroxylradicals (Singh et al., 2000). GmDREB3 transgenic tobacco accu-mulated higher levels of free proline under drought stress thanwild-type tobacco (Chen et al., 2009). Here, a quick significant(P < 0.05) increase in the proline content (Fig. 3B, Fig. 5B) inferredthat control plants might suffer a quicker and much severe osmoticstress than transgenic plants during short term drought/salinitystress and can tolerate short term drought/salinity to some extend,but not for prolonged drought/salinity stress, instead a stable pro-line content was observed during prolonged stresses. Since thefresh leaves of same weight were sampled, a significant (P < 0.05)increase in the proline content in control plant at the end of droughtstress might due to severe water loss rather than a change of phys-iological significance. In contrast, no drastic increase in prolinecontent in transgenic plants during short term stresses might beresulted from better water balance. Under prolonged stress, thetransgenic lines can enhance more proline contents to alleviatedrought and stress derived osmotic stress, which are consistentwith the finding in AtDREB1 transgenic chrysanthemum (Honget al., 2006) and TsCBF1 transgenic maize (Zhang et al., 2010), andGmDREB3 transgenic tobacco (Chen et al., 2009).

Many environmental stresses trigger the production of reac-tive oxygen species, which disrupt normal metabolism as a resultof the oxidative damage they cause to membrane lipids, proteinsand nucleic acids (Shao et al., 2008; Ashraf, 2009; Yin et al., 2009;Foyer and Shigeoka, 2011). As SOD and POD (among other so-called scavengers) are able to eliminate these harmful molecules

(Sairam et al., 2005; Yin et al., 2009), an aspect of stress tol-erance is dependent on the activation of scavengers. In presentstudy, the over-expression of CgDREBa does appear to enhancethe activity of SOD and POD under drought and salinity stress,

S. Chen et al. / Environmental and Experim

Fig. 5. Effect of salinity stress on POD activity and proline and MDA content of con-trol (WT) and transgenic (35S:TDa4 and 35S:TDa5) plants. A: POD activity. B: prolinect

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ontent. C: MDA content. * represents significant differences (p < 0.05) between theransgenic (35S:TDa4 and 35S:TDa5) plants and WT plants at the same time.

howing both short-term and long-term temporal response pat-erns, especially during prolonged stress episodes. In AtDREB1ransgenic chrysanthemum, similar change pattern in SOD activityas observed (Hong et al., 2006), suggesting these two enzymesight rather sensitive to the changes in levels of ROS during stress

uration. Cell membrane stability was estimated by lipid peroxida-ion by MDA levels. Under drought/salinity stress, MDA content ofhe transgenic chrysanthemum plants was significantly (P < 0.05)ower than in non-transgenic plants throughout the drought andalinity stress (Fig. 3C, Fig. 5C), indicating that over-expression ofhe CgDREBa gene protected the cell membrane from damage byrought/salinity stress.

In a conclusion, CgDREBa appears to function as a major tran-

criptional activator of genes within the oxidative and osmoticomeostasis pathways, it represents a promising candidate for aiotechnological approach to improving the level of crop abiotictress tolerance.

ental Botany 74 (2011) 255– 260 259

Acknowledgements

This work was supported by the Program for Hi-Tech Research,Jiangsu, China, (grant no. BG2007315, BE2008302), the NationalNatural Science Foundation of China (grant no. 30872064) andthe Fundamental Research Funds for the Central Universities (KYJ200907, KYZ201112), The Program for New Century Excellent Tal-ents in University of Chinese Ministry of Education (grant no.NCET-10-0492).

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