Identification of Flooding-Response Genes in EggplantRoots by Suppression Subtractive Hybridization

Kuan-Hung Lin & Chun-Heng Lin & Ming-Tsir Chan &

Hsiao-Feng Lo

Published online: 19 September 2009# Springer-Verlag 2009

Abstract Roots of eggplant (Solanum melongena L.) cv.Pingtong Long Eggplant (EG117) were subjected to tenflooding treatments. Messenger RNAs from plants sub-jected to flooding periods for 0.25 to 1 and 3 to 12 h as wellas unflooded plants (control) were used as the tester anddriver samples, respectively. Following suppression sub-tractive hybridization, polymerase chain reaction (PCR)products were ligated into a cloned vector for Escherichiacoli-competent cell transformation, followed by nested PCRscreening of positive cDNA inserts. PCR products of theseunique clones were digested with Sau3AI to identify anydifferentially displayed banding patterns. RecombinantcDNA clones were then picked for plasmid extraction andthen sequenced. Using BLAST and Gene Ontology data-base searches, 25 of these unigenes were found to beinvolved in metabolism, regulation, stress, and develop-ment categories, whereas nine novel genes with unknownfunctions that may have potential roles in flooding stress ineggplant were also identified. Five differentially expressedgene transcripts with known functions were randomlyselected for real-time reverse-transcription quantitativePCR to investigate plants subjected to flooding stress from0 to 72 h. All genes were upregulated in flood treatments(15 min to 72 h) when compared to the control. These

findings indicated that flood-induced genes were closelyrelated to various metabolic pathways and involved ingenetic regulation of flood stress response.

Keywords Suppression subtractive hybridization . Geneexpression . Real-time PCR . Flooding stress . Eggplant

Introduction

To expand areas of food production, crops are grown understress environments that likely contribute to lower yields.The main contributing factors to reduction in yield andquality in these areas are variations in climatic conditionssuch as flooding caused by rains (Lin et al. 2006). Heavyrainstorms and standing water can leave soils saturated fordays before draining, making waterlogging a problem inmany parts of the world. Air pockets in soil become filledwith water during saturation, thus creating hypoxic con-ditions followed by anoxia. When roots are submerged,anoxic conditions inhibit aerobic respiration, yielding lowenergy, thereby roots transport less amounts of water toshoots. Enzymes, hormones, and other solutes moving intoshoots via the transpiration stream may also be changed(Fukao and Bailey-Serres 2008; Ahmed et al. 2002). Thesechanges may constitute physiologically active messagesthat modify shoot physiology and development. Thesemodifications include chlorophyll breakdown, lower mem-brane permeability, peroxidation, slower leaf expansion,petiole epinasty, and stomatal closure (Shiono et al. 2008;Biemelt et al. 1998). Stomatal closure causes a decrease ininternal CO2 levels. Subsequently, a concomitant decline inphotosynthesis resulted from the diminished availability ofCO2 for carbon fixation, leading to senescence and evendeath of plants (Bailey-Serres and Voesenek 2008).

K.-H. Lin : C.-H. LinGraduate Institute of Biotechnology, Chinese Culture University,Taipei 111, Taiwan, Republic of China

M.-T. ChanAgricultural Biotechnology Research Center, Academia Sinica,Taipei 115, Taiwan, Republic of China

H.-F. Lo (*)Department of Horticulture, National Taiwan University,Taipei 106, Taiwan, Republic of Chinae-mail: hflo@ntu.edu.tw

Plant Mol Biol Rep (2010) 28:212–221DOI 10.1007/s11105-009-0142-z

Flooding from heavy rainfall is a major risk to freshmarket tomato production in Taiwan. Most tomato cultivarsare not capable of tolerating flooding stress. To overcomethis problem, some tomato growers use tomato seedlingsgrafted onto selected eggplant (Solanum melongena L.)rootstocks to reduce loss of tomatoes in hot wet summerseasons in Taiwan.

Previously, “Pingtong Long Eggplant” (EG117) is found tobe more flood tolerant than other cultivars and is often used asa rootstock for propagating tomatoes (Lin et al. 2004). It isreported that increased ascorbate peroxidase (APX) activitycontribute to increased waterlogged stress tolerance in theseroots. To survive flooding stress, plants have evolved anumber of physiological and biochemical responses (Perataand Alpi 1993). It has been reported that stress tolerance isthe result of a complex cascade of molecular events thatinclude gene activation and/or upregulation of stress-inducedgenes contributing to stress tolerance (Wang et al. 2007;Zheng et al. 2004; Chaves et al. 2003). Therefore, it isimportant to investigate gene expression patterns in eggplantroots to identify genes encoding proteins with regulatoryfunctions or metabolism in response to flooding conditions.

To identify and isolate those target genes that aredifferentially expressed, various protocols, such as messen-ger RNA (mRNA) differential display polymerase chainreaction (PCR; Liang and Pardee 1992), representationaldifference analysis (Hubank and Schatz 1994), microarrays(Chee et al. 1996), and suppression subtractive hybridiza-tion (SSH; Diatchenko et al. 1996), have been developed.SSH is a powerful tool for identifying abundant differen-tially expressed genes and for enriching genes of low levelsof expression (Diatchenko et al. 1996). SSH has been usedto detect differentially expressed genes in plant tissuesduring various stages of development or in response tostress (Chu et al. 2004; Mahalingam et al. 2003; Watt 2003;Hinderhofer and Zentgraf 2001).

In this study, transcripts of differentially expressed genesfrom roots of eggplants grown under two flooding stressconditions were selected by SSH. Related flood-induced genetranscripts were identified based on their putative functions.Expression of several of these differentially regulated geneswas investigated using reverse-transcription quantitative PCR(RT-qPCR). These transcriptional profiles may provideimportant insights into the regulatory pathways underlyingresponses to flooding and to other biological processes.

Materials and Methods

Plant Material, Cultivation, and Flooding Treatment

Seeds of eggplant (S. melongena L.) cv. EG117 were sown inflats in July 2006 and grown in the greenhouse as previously

described (Lin et al. 2004). After 40 days, seedlings weretransplanted to 15.4-cm-diameter plastic pots containing acommercial potting soil mix, moved to a growth chamber, andgrown under a 14-h photoperiod with an irradiance of400 μmol m−2s−1 at 35/25°C (day/night) for a period of3 days. Then pots were divided into control group, receivingno flooding treatment, and flooding treatment group, whereinplants were subjected to six flooding treatments for periods of0.25, 0.5, 1, 3, 6, and 12 h. For each treatment, threereplications were used. Pots were randomly placed in 28×14×14-cm plastic buckets and subjected to flooding by fillingthese buckets with tap water to 5 cm above the soil surface. Atdifferent time points following flooding, pots were removedfrom these buckets, and plants were removed and their rootsrinsed with tap water. Roots from each plant were clipped,placed in an icebox, moved to the laboratory within <5 min,and immediately frozen in liquid nitrogen. These were storedin a −70°C ultrafreezer until used.

RNA Isolation

Total RNA was isolated from 0.1 g of root tissues using aQiagen RNeasy plant Mini-Kit (Valencia, CA, USA) andcleaned with RNeasy reagent (Qiagen). Poly (A)+ mRNAwas extracted from total RNA with a Qiagen OligotexMini-Kit according to the vendor’s instructions. Theabsence of DNA contamination was verified by PCR usingubiquitin primers (Hoffman et al. 1991). Concentrations oftotal RNA and mRNA were determined by a Nano-DropND-1000 spectrophotometer (Nano-Drop Technologies,Paris, France) at 260 nm.

Construction of an SSH cDNA Library

SSH was performed using the “PCR Select cDNASubtraction Kit” from Clontech (San Diego, CA, USA).The SSH cDNA library was constructed according to themanufacturer’s instructions. Two mixed mRNA sampleswith equal amounts of RNA from roots subjected to 0.25-,0.5-, and 1-h (short-term) flooding treatment and to 3-, 6-,and 12-h (midterm) flooding treatment were designated as“tester S” and “tester M” cDNA pools, respectively. mRNAfrom nonflooding treatment (0 h, control) was used as the“driver.” cDNA was digested with RsaI, extracted withphenol/chloroform, precipitated with ethanol, and resus-pended in water. The digested tester cDNAwas ligated withdifferent adaptors provided in the cDNA subtraction kit.Two rounds of hybridization and PCR amplification wereconducted. The final PCR products were purified with a HighPure Product Purification Kit (Roche, Basel, Switzerland),cloned into the yT&A vector (Yeastern Biotech. Inc., Taipei,Taiwan) and transformed into Escherichia coli DH5α-competent cells.

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PCR Screening of cDNA Inserts

Subtracted cDNA clones were then plated onto LB platescontaining 50 μg/ml ampicillin, 0.4% 5-bromo-4-chloro-3-indolyl-B-D-galactosidase, and 0.1 mM isopropyl-B-D-thio-galactopyranoside. A total of 2,100 white colonies wererandomly picked and grown overnight in 96-well plates with200 μl LB/ampicillin broth. A 25-μl PCR mixture contained1-μl bacterial culture (as templates), 1 μl of each of nestedprimers 1 and 2R (10 μM of each as provided in the PCRSelect cDNA Subtractive Kit), 2.5 μl 10× Hi-Taq buffer,0.5 μl dNTP mix (10 mM), 0.2 μl (1 U) Taq DNApolymerase (Promega, Madison, WI, USA), and 18.8 μl ofH2O. PCR amplification was conducted using the followingregime: denaturation at 94°C for 3 min, followed by 30cycles of 94°C for 10 s, 68°C for 3 min, and 72°C for1.5 min and followed by a final extension at 72°C for 5 min.PCR products were analyzed on 1% agarose/ethidiumbromide gels. Of 2,100 cDNA colonies, 652 were found tobe unique in size, ranging from 0.5 to 2.5 kb. To reducefalse-positive clones in the forward SSH libraries, PCRproducts of these 652 unique inserts were then furtherdigested with Sau3AI. A total of 5 μl of each amplicon wasincubated with 1 μl Sau3AI (New England Biolab, Ipswich,MA, USA) at 37°C for 1 h, followed by electrophoreticseparation on 1% agarose gels to identify any differentiallydisplayed cDNA banding patterns. In total, 216 positivecolonies containing unique cDNA restriction maps werefinally selected, cultured in LB broth, and DNAwas isolatedusing a Plasmid Midi-Kit (Qiagen, Hilden, Germany).

DNA Sequence Analysis and BLAST Search

Those 216 differentially expressed clones were sequencedin an automated sequencer, ABI Prism 3100 (Perkin ElmerABD, CA, USA), using universal forward or reverseprimers homologous to the vector sequence (BioengineerCo., Taipei, Taiwan). Sequences were compared to those innonredundant protein database in GenBank using theBLASTX and BLASTN programs.

cDNAs with BLASTX E values of ≤1×10−10 weredesignated as having significant homology. The putativephysiological functional categories of the identified geneswere assigned based on GoFigure of Gene Ontologyannotations (http://www.geneontology.org).

RT-qPCR and Quantification of RNA Levels

Total RNA was extracted from roots of eggplants subjectedto ten flooding treatments (0, 0.25, 0.5, 1, 3, 6, 12, 24, 48,and 72 h) as described above. The first-strand cDNAsynthesis was performed with an Oligo (dT) primer usingRETROscript Reverse Transcription for the RT-PCR Kit

(Ambion, Austin, TX, USA). cDNA was diluted 1:5 forreal-time PCRs which were carried out in 384-well plates ina Light Cycler 480 (Roche, Basel, Switzerland). Five gene-specific primer pairs, from the above-mentioned sequencedgenes for RT-qPCR, were designed using Light CyclerProbe design software (Roche) and synthesized commer-cially (Bioengineer Co.). Each RT-qPCR reaction (20 μl)contained 1 μl diluted cDNA, 9 μl ddH2O, 2 μl Primer Mix(10 ×), 4 μl 5× SYBR green I, 1.6 μl MgCl2, 0.4 μl of theenzyme mix, and 2-μl resolution solution (Roche RNAAmplification Kit). The amplification program consisted ofone cycle of 95°C for 5 min for preincubation, followed by40 cycles of 95°C for 30 s, 58°C for 10 s, and 72°C for20 s. After amplification, a melting curve analysis was runusing the program of one cycle at 95°C for 5 s, 65°C for10 s, and 95°C with 0 s held in the step acquisition mode,followed by cooling at 40°C for 30 s.

A total of three replicates were performed for each cDNAsample, and template-free and negative controls were set. Tonormalize the total amount of cDNA in each reaction, theubiquitin gene (Hoffman et al. 1991) was coamplified as aninternal control. Data were analyzed with the PCR efficiencycorrection using Light Cycler 480 Relative Quantificationsoftware V1.01 (Roche) based on relative standard curvesdescribing PCR efficiencies of target and reference genes.

Results

Identification and Sequencing of Differentially ExpressedGene Transcripts by SSH

Two flooding-induced SSH libraries of eggplant roots wereconstructed using two flooding treatments as testers and anonflooding treatment as the driver. Both short-period (S)and moderate-period (M) SSH libraries were likelyenriched in cDNAs representing genes preferentiallyexpressed during different flooding periods. In total, 1,050clones from each library were randomly picked for PCRscreening. Of these, 268 (25.5%) of the “S” library and 384(36.6%) of the “M” library contained differentiallyexpressed cDNA inserts, whereas the remaining clones(74.5% of “S” and 63.4% of “M”) showed either low or nodifferences in band intensities and were considered to befalse-positive clones (data not shown). Amplicons fromthese positive similar-sized clones were digested withSau3AI and analyzed by gel electrophoresis to confirmpresence of unique clone inserts. The fragments rangedfrom 0.4 to 3 kb with most fragments distributed between0.7 and 1.5 kb (Fig. 1). Eighty-two of 268 clones (from the“S” library) and 134 of 384 clones (from the “M” library)were deemed unique differentially expressed gene frag-ments (Fig. 1) and were subjected to sequencing.

214 Plant Mol Biol Rep (2010) 28:212–221

Functional Analysis and Characterization of the SSHLibraries

Identified sequences generated from “S” and “M” librarieswere analyzed separately by BLAST, classified based onputative functions using the Gene Ontology hierarchy, andgrouped into two (Table 1) and four (Table 2) classes,respectively. Eighty-two cDNAs had high sequence simi-larity (> 88% identity, 0~E−37) to database entries,suggesting that they were either the same gene or belongedto the same gene family. Of 82 gene clusters, only threestress-responsive genes were found to be involved inmetabolism (clone A14) and stress (clones I44 and I17)under short-term flooding stress conditions (Table 1). Of134 transcript clusters, 31 stress-responsive genes exhibitedfunctions related to metabolism, stress, regulation, devel-opment, and unknown functions under moderate-lengthflooding treatments (Table 2). These 31 annotations of blastsequences had various ranges of similarity with matchesfrom 80% (E−20, clone FC67) to 100% (0, clones FC43,FC60, A80, and SA45), and sequenced lengths ranged from120 (clone SA63) to 1,000 bp (clone FB15). Interestingly,the majority of these unknown novel genes were fromtomato clones (Table 2).

Confirmation of Flooding-Regulated Differential GeneExpression

Five gene-specific primer pairs (forward and reverse pairs)of expected amplification sizes for selected transcripts arelisted in Table 3. These are randomly selected from the “M”library and consist of the following: a gene (SC22) formetabolism, two genes (FC22 and FC43) for stress, andtwo genes (FC60 and SA28) for regulation (Table 2). RT-qPCR analyses of EG117 roots subjected to flooding from0 to 72 h are shown (Fig. 2). All data are normalized toRNA levels of the housekeeping gene ubiquitin.

RNA expression of abscisic acid (ABA) 8′-hydroxylaseincreased from 15 (1.44) to 30 min (3.07) of flooding,dropped to 1.84 at 60 min of flooding, peaked (4.41) after12 h of flooding treatment, and dropped thereafter (Fig. 2a).The transcript of the HSR201 gene coding for X95343initially increased up to 6 h of flooding (7.52) and thendecreased to 6.44 at 12 h of flooding. At 24 h of flooding,the level peaked at 6.73 and began to drop thereafter(Fig. 2b). Patterns and levels of alcohol dehydrogenaseRNA expression varied over time (Fig. 2c). Levels oftranscripts, dramatically changed relative to expressionlevels at the peaks, and they accumulated at different rates

Fig. 1 Twenty-four PCR prod-ucts from suppression subtrac-tive hybridization were digestedwith Sau3AI and electrophor-esed to confirm unique cloneinserts (circle) for DNAsequencing

Table 1 Proposed identities of overexpressed genes of eggplant root suppression subtractive hybridization (SSH) library from short-term flooding(0.25 to1 h) and their functional classification following BLAST

Clone cDNA (bp) Homology sequence E value Sequenceidentity (%)

Accession no.

Metabolism

A14 228 Malus x domestica NADP-dependent malic protein-like mRNA 0 100 AY279309

Stress

144 457 Populus euphratica clone PSR1 salt-induced mRNA 0 100 AF315118

117 457 Capsicum chinense pathogenesis-related (PR) 4b-Cc mRNA for PR protein 6E−37 88 AB162223

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from 0.25 (2.10) to 72 h (13.75) of flooding. Transcripts ofthe G/HBF-1 gene, encoding for Y10685, also changedduring flooding. The highest increase (385.22) in tran-scripts was detected at 6 h of flooding and was 77-foldhigher than that at 1 h of flooding (4.99; Fig. 2d).Transcripts of the mitogen-activated protein (MAP) kinase

gene showed a rapid increase at 3 h (110.67), decreased to15.42 at 6 h, and remained low thereafter (Fig. 2e).

Overall, three patterns of expression were observedfollowing flooding. These include peaks at 6 and 24 h(Figs. 2b–d), peaks at 0.5 and 12 h (Fig. 2a), and a peakonly at 3 h (Fig. 2e). All target genes (Fig. 2a–e) were

Table 2 Proposed identities of overexpressed genes of eggplant root suppression subtractive hybridization (SSH) library from midterm flooding(3 to 12 h) and their functional classification following BLAST

Clone cDNA (bp) Homologysequence

E value Sequenceidentity (%)

Accessionno.

Metabolism

SC22a 477 Solanum tuberosum ABA 8'-hydroxylase CYP707A1 mRNA 8E−169 93 DQ206630

FA87 127 Tomato H+-ATPase (LHA1) mRNA 9E−39 96 M60166

FB61 180 Lycopersicon esculentum beta-galactosidase (TBG3) mRNA 6E−44 94 AF154421

FC85 518 Solanum tuberosum subsp. tuberosum mRNA for sucrose synthase 2E−101 85 AJ537575

SA63 120 Arabidopsis thaliana trehalose-phosphatase AT1G23870 (ATTPS9) mRNA 7E−18 88 NM102235

A33 301 Nicotiana tabacum mRNA for inorganic pyrophosphatase (PPase) 6E−101 93 X83729

A63 349 Nicotiana tabacum mRNA for glyceraldehydes-3-phosphatedehydrogenase

3E−124 93 AJ133422

D24 229 Solanum chacoense putative G-protein-coupled receptor mRNA 2E−10 83 AF272710

Stress

FC22a 760 Nicotiana tabacum mRNA for hypersensitive response (HSR)201 harpin-induced spermine protein

3E−169 85 X95343

FB88 206 Solanum berthaultii thioredoxin mRNA 7E−10 86 DQ413184

FA80 412 Capsicum annuum ethylene-responsive factor-like protein 1 (ERFLP1) 7E−18 84 AY529642

FC43a 529 Arachis hypogaea clone Gsi115 alcohol dehydrogenase (Adh) mRNA 0 100 AY725189

A7 300 Solanum pheja cultivar 1-3/84 putative heat shock protein mRNA 1E−98 92 AY573846

A15 620 Nicotiana tabacum HtHSF2 mRNA for heat shock factor 3E−67 86 AB014484

Regulation

FC60a 795 G. max mRNA for G-box and H-box binding factor (G/HBF)-1 0 100 Y10685

FC67 890 Arabidopsis thaliana transcription factor AT1G08620 mRNA 6E−20 80 NM100735

SA28a 453 Capsicum annuum mitogen-activated protein (MAP) kinase1 (MK1) mRNA

1E−179 94 AF247135

A80 796 Potato hydroxyl-3-methylglutaryl coenzyme A reductase (hmgr) mRNA 0 100 L01400

C43 492 Nicotiana glauca putative delta tonoplast intrinsic protein (TIP)MIP2 mRNA

9E−58 82 AF290618

C33 388 Lycopersicon esculentum regulator of gene silencing mRNA 1E−114 90 AY642285

C90 529 Solanum tuberosum DNA-binding protein homolog mRNA 2E−25 87 U77655

Development

SB32 675 Nicotiana tabacum A20 mRNA for hypothetical protein 0 100 AB032535

Unknown gene

SA11 143 Lycopersicon esculentum clone 114253R mRNA 6E−31 90 BT013015

SA8 428 Lycopersicon esculentum clone 133928F mRNA 2E−178 95 BT014523

A23 195 Lycopersicon esculentum clone 132757F mRNA 1E−23 98 BT013828

B70 183 Lycopersicon esculentum clone 134032F mRNA 3E−58 95 BT014577

D10 451 Lycopersicon esculentum clone 134376R mRNA 2E−172 95 BT013203

C31 340 Lycopersicon esculentum clone 133830F mRNA 3E−19 81 BT014479

FB15 1,000 Qryza sativa (japonica cultivar group), predicted mRNA 1E−12 81 NM193615

SA56 515 Solanum tuberosum clone 147D03 DnaJ-like protein mRNA 7E−151 91 DQ228340

FA5 180 Solanum tuberosum clone 134A04 POM30-like protein mRNA 1E−12 91 DQ22832

a Sequences selected for RT-qPCR

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upregulated from 0.25 to 72 h of flooding, when comparedto control (0 min).

Discussion

In this study, different stages of flooding stress in roottissues of eggplant were investigated. In particular, tran-scriptional responses of eggplant to both short-term andmidterm flooding stress were determined. Analysis ofdifferentially expressed genes revealed that 82 of 268clones (30.6%) were from the early-stage flooding SSHlibrary, and 134 of 384 clones (34.9%) were from themidterm-stage flooding SSH library. With longer floodingperiods, addition clones were identified. In total, 4.3%unique genes were detected, and these were highlyupregulated by mild (3 to 12 h) flooding stress. Thesemidterm response genes were probably critical for activat-ing and integrating stress defense mechanisms and thusplayed important roles in the plant’s defense againstflooding stress. Kawasaki et al. 2001 identified a set ofnew early response genes in rice grown under salt stress for30 min and found it to be associated with salt stress. Theseearly response genes were reported to be stress sensors ofinducible transcriptional activators or upstream signalpathway components and thus might act as fate determi-nators of salt tolerance. In this study, a set of five regulatedcDNAs, of known functions, was selected from themidresponse library for relative expression analysis byRT-qPCR during various flood durations. Therefore, thesemidterm response genes might also serve as determinants offlood tolerance in eggplants.

Flooding stress has a harmful effect on overall plantgrowth, affecting leaves, flowers, and roots. In this study,no strong visible effects were observed on the leaves,flowers, or roots of eggplants in response to early flooding.After 48 h of exposure, some plants exhibited stresssymptoms, such as epinasty, necrosis, and senescence

(i.e., chlorosis) in some leaves and flowers, and the rootapex appeared brownish in color (data not shown). After72 h of flooding stress, most plants appeared epinastic withwilting and loss of photosynthetic capability, and thesecharacteristics did not disappear after plants were removedfrom stress conditions. In general, early flooding had noharmful effects on plants; yet, with prolonged floodingstress, significant flooding injury was observed. The flood-ing stress conditions used in this study (0 to 72 h)influenced plant growth, but these effects were not lethal.Thus, observed responses of eggplants probably includedadaptive responses to flooding.

In total, 216 gene transcripts of different levels ofexpression in the two flooding treatments used in this studyhave been identified, including genes encoding proteinsinvolved in metabolism, regulation, development, andstress-related conditions. This clearly suggests functionaldifferentiation of flooding periods. Some of these genes areinvolved in common molecular mechanisms (e.g., metabol-ic pathways, regulatory networks, genetic development, andstress-related proteins) in response to flood stress condi-tions, and these genes must be involved in both plantdevelopment and stress response. In total, nine genes in the“metabolism” category encode different enzymatic func-tions, such as hydroxylase, galactosidase, sucrose synthase,phosphatase, pyrophosphatase, and dehydrogenase, amongothers. These suggest that a wide range of biochemicalactivities may be involved in flooding stress. In addition,the transcriptional activation of various metabolic pathwaygenes can be an important step in regulating the accumu-lation of aforementioned proteins during a plant’s responseto flooding stress. It is important to determine the functionsand interactions of genes involved in molecular mecha-nisms mediating flood acclimation. The acclimation processis reported to involve new proteins synthesized in responseto flood stress. Eight stress-related genes, PSR1, PR4b-Cc,HSR201, thioredoxin, ERFLP1, Adh, HSP, and HSF, havebeen identified in this study. These genes may play

Table 3 Primers used for amplification of transcripts in eggplant by RT-qPCR

Clone ID Forward and reverse primers(5′→3′) Accession number and the expected amplification size

ABA 8'-hydroxylase TCCTACATGCTGTCACAGA FJ842384 (435 bp)ATGATGGACCCCATTG

HSR201 protein AAACCCGATCCTGAAGAG FJ860276 (740 bp)ACTTGGTCGAATCCCG

Alcohol dehydrogenase AATGGGTGCTGGTCT FJ860278 (487 bp)AATGACTCGTCCTCCG

G/HBF-1 CCACGGATACGCTTCT FJ860277 (803 bp)CCAACAACCGCCAAAT

MAP kinase 1 GAATCGTCTGCTCGGT FJ860279 (411 bp)GCAGGTACTGGCAGTGA

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Fig. 2 Real-time PCR of a ABA 8′-hydroxylase mRNA, b HSR201protein mRNA, c alcohol dehydrogenase mRNA, d G/HBF-1 mRNA,and e MAP kinase 1 mRNA. These correspond to response from roots

of eggplant cv. EG117 exposed to 0 to 72 h of flooding. Relativeamounts were calculated and normalized with respect to ubiquitinmRNA

218 Plant Mol Biol Rep (2010) 28:212–221

important roles in crosstalk in different forms of abioticstress and signaling pathways (Ouyang et al. 2007).Acclimation to flood stress involves the coordinatedengagement of regulatory networks. Seven genes, includingG/HBF-1, transcription factor, MAP kinase, hydroxyme-thylglutaryl coenzyme A reductase, tonoplast intrinsicprotein, gene silencing regulator, and DNA-binding protein,are known as members of the “regulation” group that areupregulated at midterm stage of flooding stress. Transcrip-tion factors usually play important roles in signal transduc-tion pathways and are the earliest group of genes to respondto abiotic stresses (Shinozaki et al. 2003). Molecular andgenomic studies have shown that various transcriptionfactors are involved in regulating stress-inducible genes(Dubouzet et al. 2003).

In this study, 31 and three genes were found to beresponsive to mild and slight flooding stress, respectively,and these belonged to four different functional groups. Thissuggested that most of the genes were not turned on at theearly stage of flooding but came on when a longer floodingstress was imposed. Under flood conditions, the plants grewslowly and occasionally suffered growth defects or damage.These flood-induced growth changes might be attributed tothese four functions. Furthermore, none of these functionswere transcribed from early responsive genes encoding bothdevelopment and regulation functions, indicating thatchanges in levels of expression of developmental andregulation genes occurred later in flooding acclimation.

Metabolism-, regulatory-, and stress-related genes werefound more frequently in the “M” library than in the “S”library (Tables 1 and 2). Therefore, five differentiallyexpressed genes from these functional categories wereselected to further explore changes in mRNA abundanceacross flooding periods. As flooding stress over timeresulted in changes in all transcripts, with the highestmRNA levels detected at 3 to 12 h of flooding (Fig. 2), thisindicated that these genes were preferentially expressed atmidterm flooding. This finding also supported the SSHresults wherein these five genes were differentially upregu-lated in response to flooding.

Saika et al. (2007) have demonstrated that the rapidlowering of ABA that occurs upon submergence of lowlandrice is due to ABA 8′-hydroxylation, which is activatedthrough ethylene-induced upregulation of OsABA8 ox1 (agene encoding ABA 8′-hydroxylase) expression, as well asthe ethylene-independent suppression of ABA biosynthesis.In this study, ABA 8′-hydroxylase mRNA began toaccumulate as early as 0.5 h following flooding, indicatingthat ethylene biosynthesis may be enhanced upon short-term flooding. Fukao and Bailey-Serres (2004) havereported that ethylene is one of the major components ofresponses to hypoxia. Reactive oxygen species productionis also a component of the pathway that induces alcohol

dehydrogenase expression under low oxygen. Ethylenesignaling under flooding may explain the observed increasein alcohol dehydrogenase mRNA in eggplant roots underflooding (Fig. 2c). Future studies should investigatewhether there is crosstalk among ethylene, ABA 8′-hydroxylase, and alcohol dehydrogenase signaling path-ways under flooding conditions. Studies of protein kinaseshave shown that MAP kinases are activated by H2O2 inplants, which could lead to the modulation of geneexpression (Zwerger and Hirt 2001; Torres and Forman2003). Activation of MAP kinases by H2O2 is a critical stepin mediating cellular responses to multiple stresses (Kovtunet al. 2000). It is likely that eggplants accumulatingexcessive amounts of MAP kinase 1 in leaves (Fig. 2e)aid in plant defense against the harmful oxidative stresscaused by flooding. The function of MAP kinases and theirinteractions with H2O2 in flooding signal transductionremain to be elucidated. In plants, various transcriptionfactors have been implicated in selective regulation of geneexpression during development and in response to environ-mental cues (Droge-Laser et al. 1997). G-box (CACGTG)and H-box (CCTACC) binding factors (G/HBF) areinvolved in light signal transduction in Arabidopsis.Meanwhile, G-box binding factor 1 with basic leucinezipper (bZIP) protein regulates ABA-induced transcription(Mallappa et al. 2006; Shiota et al. 2008). In this study, G/HBF1 acts as a positive regulator and is differentiallyregulated from during the 72 h of flooding. This demon-strates that G/HBF1 may be a transcription factor ineggplants for rapid and flexible regulation of selective geneexpression by flooding stimuli.

In this study, results showed that a group of stress-relatedgenes was induced under flood conditions. In contrast, themore widely investigated antioxidant genes, such as theAPXs and SODs (Lin et al. 2007; Watkinson et al. 2006),did not respond to flood stress in this study. This wasprobably due to the limited number of clones that weresequenced, differences in the timing and method ofselection, and/or differences between the “S” and “M”libraries used for screening. In some cases, sequences wereshort and could not be annotated. Wu et al. (2008) utilized acombination of SSH, microarray, and real-time RT-PCR toidentify 36 genes differentially expressed in CS-B22shcotton 15-dpa fibers. Using SSH, Zeng et al. (2006)identified 671 differentially expressed cDNAs in cottonsomatic embryos, and more than one third of those cDNAshad not been reported in the GenBank databases. Zhang etal. (2005) indicated that about 30% of clones in an SSHlibrary had no similarity to sequences in the database. Ninemidterm flood-response genes regulated by flood stress inthis study did not have significant homologies to cDNAswith assigned functions for products and encoded unknownproteins. This suggested that a large proportion of upregu-

Plant Mol Biol Rep (2010) 28:212–221 219

lated genes in eggplant roots were unique. Furthercharacterization and functional analysis of these geneswould facilitate our understanding of the flood-responsemechanism in eggplant.

Acknowledgements This research was supported by grants from theNational Science Council, Taiwan, ROC. The authors are grateful toMs. Shu-Yen Pi for typing and editing this manuscript.

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