Ethylene Response Factor TERF1 Enhances Glucose Sensitivity in Tobacco through Activating the Expression of Sugar-related Genes

Journal of Integrative Plant Biology 2009, 51 (2): 184–193

Ethylene Response Factor TERF1 Enhances GlucoseSensitivity in Tobacco through Activating the Expression

of Sugar-related Genes

Ang Li1,2, Zhijin Zhang2,3,4, Xue-Chen Wang1 and Rongfeng Huang2,3,4∗

(1National Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China;2Biotechnology Research Institute, the Chinese Academy of Agricultural Sciences, Beijing 100081, China;

3National Key Facility of Crop Gene Resources and Genetic Improvement, Beijing 100081, China;4National Center for Plant Gene Research (Beijing), Beijing 100081, China)

Abstract

Ethylene response factor (ERF) proteins are important plant-specific transcription factors. Increasing evidence showsthat ERF proteins regulate plant pathogen resistance, abiotic stress response and plant development through interactionwith different stress responsive pathways. Previously, we revealed that overexpression of TERF1 in tobacco activatesa cluster gene expression through interacting with GCC box and dehydration responsive element (DRE), resulting inenhanced sensitivity to abscisic acid (ABA) and tolerance to drought, and dark green leaves of mature plants, indicatingthat TERF1 participates in the integration of ethylene and osmotic responses. Here we further report that overexpressionof TERF1 confers sugar response in tobacco. Analysis of the novel isolated tomato TERF1 promoter provides informationindicating that there are many cis-acting elements, including sugar responsive elements (SURE) and W box, suggesting thatTERF1 might be sugar inducible. This prediction is confirmed by results of reverse transcription-polymerase chain reactionamplification, indicating that transcripts of TERF1 are accumulated in tomato seedlings after application of glucose. Furtherinvestigation indicates that the expression of TERF1 in tobacco enhances sensitivity to glucose during seed germination,root and seedling development, showing a decrease of the fresh weight and root elongation under glucose treatment.Detailed investigations provide evidence that TERF1 interacts with the sugar responsive cis-acting element SURE andactivates the expression of sugar response genes, establishing the transcriptional regulation of TERF1 in sugar response.Therefore, our results deepen our understanding of the glucose response mediated by the ERF protein TERF1 in tobacco.

Key words: Ethylene response factor protein TERF1; glucose; sugar responsive gene; tobacco.

Li A, Zhang Z, Wang XC, Huang R (2009). Ethylene response factor TERF1 enhances glucose sensitivity in tobacco through activating the expressionof sugar-related genes. J. Integr. Plant Biol. 51(2), 184–193.

Available online at www.jipb.net

Sugars, as the prime carbon and energy sources, functionas metabolic resources and structural constituents of most celltypes (Coruzzi and Zhou 2001; Finkelstein and Gibson 2002;Baena-Gonza′lez et al. 2007). During the life cycle of plants,

Received 12 Aug. 2008 Accepted 7 Oct. 2008

Supported by the National Natural Science Foundation of China (30525034)

and the State Key Basic Research and Development Plan of China

(2006CB100102).∗Author for correspondence.

Tel: +86 10 6213 9060;

Fax: +86 10 8210 6142;

E-mail: <[email protected]>.

C© 2008 Institute of Botany, the Chinese Academy of Sciences

doi: 10.1111/j.1744-7909.2008.00794.x

sugars modulate various processes such as embryogenesis,seed germination, seedling development, floral transition, fruitripening, and senescence (Paul and Pellny 2003; Borisjuk et al.2004; Lu et al. 2007). Most importantly, as signal molecules,sugars have been identified to be involved in these physiolog-ical processes through the regulation of transcription factors(Koussevitzky et al. 2007; Lu et al. 2007; Hanson et al. 2008).Hexokinase (HXK), the first enzyme in glycolysis, has beenestablished to be a sensor of glucose response (Jang et al.1997; Zhou et al. 1998; Moore et al. 2003). Moreover, theHXK1-dependent pathway is affected by abscisic acid (ABA)biosynthesis and signaling components. Glucose-insensitive(gin) mutants have been proved to be allelic to ABA-insensitive(abi) mutants, ABA-deficient mutants (aba1–1, aba2–1, aba3–2) are also glucose-insensitive mutants. For instance, gin1mutant is allelic to aba2 (Laby et al. 2000; Rook et al. 2001), and

Regulation of TERF1 in Sugar Response 185

ABA-insensitive mutants abi4 and abi5 exhibit the gin phenotype(Arroyo et al. 2003). It has been revealed that glucose playsan important role in the basal level of ABA, via regulating theexpression of ABA2, ABA1 and ABA3; while glucose-insensitivemutants gin1 and gin5 could be restored by ABA (Arenas-Huertero et al. 2000; Cheng et al. 2002). With the understandingof glucose signaling, it is further realized that components of theglucose pathway participate in ethylene biosynthesis and thesignaling pathway as well.

Ethylene is the simplest gaseous phytohormone that mod-ulates developmental processes and responses to stressand pathogen attack (Johnson and Ecker 1998; Stepanovaand Alonso 2005). The production of ethylene starts fromthe conversion of S-adenosyl-L-methionine (AdoMet) to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase.ACC is the immediate precursor of ethylene, and can becatalyzed to ethylene by ACC oxidase (Wang et al. 2002). InArabidopsis, ethylene is perceived by a family of membranereceptors, ETR1, ERS1, ETR2, EIN4, and ERS2 (Hua et al.1995, 1998; Sakai et al. 1998), which result in the deactivation ofCTR1 and the activation of the positive regulator EIN2 (Alonsoet al. 1999). EIN2 further regulates EIN3 (Chao et al. 1997),which binds to the promoters of genes encoding ethyleneresponse factor (ERF) genes, such as ethylene responsivefactor 1 (ERF1). The ERF1 and other ERF proteins interact withthe GCC box in the promoters of target genes and then initiatea transcriptional cascade leading to activation of downstreamethylene responses (Chao et al. 1997; Solano et al. 1998).The involvement of ethylene-induced phenotypes with small,dark-green rosettes was observed in the mutants of constitutiveethylene response (ctr1) and ethylene overproducer (eto1),and gin1 as well (Zhou et al. 1998). The glucose-insensitivephenotype could be mimicked by treatment of wild-type plantswith ethylene precursor ACC (Zhou et al. 1998; Leon and Sheen2003). Current research has demonstrated that constitutiveethylene biosynthesis and signaling mutants show insensitivityto glucose (Arroyo et al. 2003; Price et al. 2004; Rollandet al. 2006). Prolonged glucose treatment revealed that gin4is allelic to ctr1, while ethylene-insensitive mutants, such asein2 and etr1-1 exhibit glucose hypersensitivity. These resultsconfirm that ethylene could overcome the glucose-dependentdevelopmental arrest (Leon and Sheen 2003). Furthermore,the finding that glucose and ethylene antagonistically regulateprotein stability of the EIN3 reveals a molecular linkage betweenglucose and ethylene signaling (Yanagisawa et al. 2003).

Previously, we have shown that a tomato ERF activatorTERF1 could bind to GCC box and dehydration responsiveelement (DRE), and that the expression of TERF1 in tobaccoinduces ethylene triple response, and results in increased ABAsensitivity and osmotic stress tolerance through activating theexpression of downstream genes (Zhang et al. 2005). Moreinterestingly, it has been proved that glucose-insensitive mutantgin1–1 displays smaller and darker-green leaves (Zhou et al.

1998), which is consistent with our observation that expressingTERF1 in tobacco shows the phenotype of dark green leaves(Huang et al. 2004). However, whether and how ERF proteinsare involved in the sugar response is not yet established. In thepresent paper, we report the involvement of ERF protein TERF1in the response to glucose in tobacco.

Results

Promoter of TERF1 contains putative sugar responsiveelements

Our previous investigations have indicated that the ERF geneTERF1 is significantly accumulated in response to ethylene,drought, cold and ABA, conferring the regulation in ethyleneand ABA responses (Huang et al. 2004; Zhang et al. 2005).Since the multiple responses of TERF1, it is reasonable toisolate and analyze the promoter of TERF1 for further studies.Through Tail-PCR (thermal asymmetric interlaced-polymerasechain reaction) using tomato genomic DNA as a template, weinitially isolated an approximately 1.4 kb fragment upstreamof the translation start site of TERF1 (Accession numberEU395634). Sequence analysis using the PLACE database(http://www.dna.affrc.go.jp/PLACE/) indicates that this fragmentcontains not only the basal elements, TATA box and CAAT box,nearing start code ATG, but also several phytohormone respon-sive cis-elements, such as ABA responsive element (ABRE,ACGTG), salicylic acid responsive element (SARE, TTGAC),auxin responsive element (AuxRE, TGTCTC), gibberellin re-sponsive element (GARE, CAACTC), and sugar responsiveelements SURE and W box (Figure 1A and Figure S1). Thepresence of ABRE that is responsible for ABA and droughtresponses is consistent with the characterization of TERF1(Zhang et al. 2005). More interestingly, the distal location ofsugar responsive elements SURE and W box suggests thepossible regulation of TERF1 in sugar response.

Next we then tested the expression of TERF1 in response toglucose and mannitol using semi-quantitative reverse transcrip-tion (RT)-PCR assays. Our results indicated that the transcriptlevels of TERF1 in tomato seedlings enhanced up to fivefoldfrom the concentration of 1% to 3%, and then decreased atthe concentration of 6%, compared with the untreated control,after incubation for 24 h treatment of glucose; while mannitoltreatment did not cause obvious changes of TERF1 transcripts(Figure 1B), indicating the possible regulation of TERF1 inglucose response.

Expression of TERF1 in tobacco increases sensitivity toglucose during seed germination, root and seedlingdevelopment

In order to investigate the involvement of TERF1 in the sugarresponse, we used a seed germination assay to test whether

186 Journal of Integrative Plant Biology Vol. 51 No. 2 2009

Figure 1. Sequence analysis of TERF1 promoter and expression of

TERF1 in response to glucose and mannitol in tomato.

(A) Deduced cis-acting elements presence in the promoter region.

(B) Expression of TERF1 in response to glucose and mannitol in tomato.

Semi-quantitative reverse transcription-polymerase chain reaction (RT-

PCR) detects the expression of TERF1 in response to glucose and

mannitol. cDNAs converted from RNA samples of 2-week-old tomato

leaves as a template of PCR amplifications. The expression levels

are relative to the control of H2O treatment that is taken as 100 after

normalizing against the actin signal. Error bars are based on three

independent experiments.

overexpression of TERF1 in tobacco (OE) alters the responseof plants to glucose. Our results indicated that seed germinationof OE lines and wild type (WT) tobacco seeds in glucose-free Murashige and Skoog (MS) medium approached a similargermination after plating seeds for 72 h. In the presence ofexogenous glucose, germination in OE lines was more sensitiveto glucose than that in WT. On 3.6% glucose plates, germinationat the fourth day was about 85% in WT, while it was only20%–28% in OE lines. This sensitivity to glucose in OE lineswas enhanced with the increase of glucose concentrations(Figure 2), suggesting that expression of TERF1 in tobaccoenhances the sensitivity to glucose during seed germination.

Next we compared the root elongation between WT and OEseedlings. After treatment with glucose for 7 d, the root lengthof seedlings reached the longest elongation at 1% of glucosecompared with that of the control (0% Glc). With the steadyincrease of glucose concentrations (3%–7%), root elongationswere arrested in both WT and OE lines, but more seriously in-hibited in OE seedlings (Figure 3A). To determine the inhibitoryextent of root elongation by glucose, we then measured the rootlengths of seedlings grown on MS medium supplemented withvarious concentrations of glucose. Compared with those in WT,the root lengths in OE seedlings were less than 50% of the wildtype at the same concentration of glucose under 6% and 7%glucose (Figure 3B). In order to confirm the above observation,

Figure 2. Expression of TERF1 in tobacco delayed seed germination

under glucose treatment.

Tobacco seeds were kept in Murashige and Skoog (MS) medium

containing glucose. Germinated seeds were recorded 4 d after plating

seeds on MS medium. About 100 seeds were used for each treatment.

Results are the average of three replicates ± SD.

we further used mannitol to exclude the effect of osmoticum onthe root elongation. Interestingly, the root lengths in WT and OElines were obviously inhibited with no difference when seedlingswere treated with 3% and 6% mannitol (Figure 3C), suggestingthat TERF1 enhances tobacco root sensitivity to glucose.

Previous studies have demonstrated that high levels of exo-genous sugars could repress seedling development, such ashypocotyl elongation, cotyledon greening and expansion, andshoot development (Rolland et al. 2006). Consistent with theabove results of Figure 3A, the seedlings reached the highestgrowth at 1% of glucose compared with that of the control (0%Glc) after glucose culturing for 10 d. Higher concentration ofglucose (3%–6%) significantly impaired seedling developmentin both WT and OE lines; while the arrest of seedling devel-opment did not have an obvious difference between WT andOE lines when germinated seedlings were grown on a mediumof 3%–6% mannitol (Figure 4A). The data from fresh weightof seedlings further supported our aforementioned observation.As shown in Figure 4B, at the same concentration of 3% and6% glucose, OE seedlings had only 70% and 50% fresh weightof WT, respectively. However, no obvious difference in freshweight between WT and OE under mannitol treatment wasfound (data not shown). These result demonstrated that theenhanced sensitivity to glucose in TERF1-expression seedlingsis unique to the effect of osmotic stress.


Figure 3. Expression of TERF1 in tobacco arrested root elongation under glucose treatment.

(A) Roots with about 1 cm length were placed vertically in Petri dishes supplemented with glucose for 7 d. The images are taken after growth

for 7 d.

(B) Relative elongation rate of seedlings on Murashige and Skoog (MS) media in the presence of glucose after growth for 7 d.

(C) Roots with about 1 cm length were placed vertically in Petri dishes supplemented with mannitol for 7 d. The images are taken after growth

for 7 d.

Data from six to seven seedings are the average of three replicates ± SD.

TERF1 activates the expression of SURE-driven reporterin transient assays

The response of expressing TERF1 in tobacco to glucosesuggests that TERF1 might regulate the expression of glucose-related genes. Although cis-elements involved in sugar signalinghave been reported, no common regulatory DNA elements areevident for sugar response (Rolland et al. 2006). The sucrose-responsive element (SURE) was first identified from sweetpotato tuber class I patatin promoter (Grierson et al. 1994). Theinvestigation of rice α-amylase gene reveals three essential cis-elements that are important for high sugar starvation-inducedexpression: the GC-box, the G-box, and the TATCCA element(Chan and Yu 1998; Hwang et al. 1998; Lu et al. 1998).Studies on sugar activation of SUS, sporamin and β-amylasepromoters have identified several cis-elements, including B-box(Grierson et al. 1994; Zourelidou et al. 2002), the TGGACGGelement (Maeo et al. 2001), and an SP8 motif (Ishiguro andNakamura 1994). The G-box motif (CACGTG) is involved inthe phytochrome-mediated control of gene expression (Giulianoet al. 1988), which is very similar to ABRE (Ohto et al. 1992).The core sequence of the WRKY binding element (W-box) is

found in the promoters of wheat, barley, and wild oat α-AMY2gene (Rushton et al. 1995). A WRKY-type SUSIBA2 is sugar-inducible and binds to the SURE and W-box (Sun et al. 2003).These results suggest that sugar and hormone signaling mightcross in the transcriptional modulation of W-box, SURE andG-boxes in various promoters. Previously, we have reportedthat TERF1 activates the transcription of GCC box- and DRE-containing genes in vivo and in vitro (Huang et al. 2004; Zhanget al. 2005). The fact that the expression of TERF1 is glucoseresponsible in tomato (Figure 1B), and that the expressionof TERF1 in tobacco exhibits glucose-sensitive phenotypes(Figures 2,3), inspired us to detect whether TERF1 binds tothe sugar response element to regulate the expression of sugarresponse genes. To reveal the transcriptional regulation, we firstinvestigated the interaction of TERF1 with sugar responsivecis-acting elements, SURE and W-box, using Agrobacterium-mediated transient assay in vivo. As shown in Figure 5, TERF1strongly activates the transcription of the reporter gene, and theactivity of the GUS gene driven by SURE was 11-fold higherthan that of the control (Min), whereas the activity driven by W-box showed no obvious difference from the control (Figure 5),indicating that TERF1 interacts with SURE but not W-box.


Figure 4. Expression of TERF1 enhances the sensitivity to glucose

during seedling development in tobacco.

(A) Seeds were germinated on Murashige and Skoog (MS) medium

supplemented with 0.6% agar. After germination, seeds were cultured

with or without glucose and mannitol for 10 d. Upper panels show

seedlings in plates, while lower panels display single seedling from upper

respective plates.

(B) Fresh weight of seedlings was measured 10 d after germination.

Results are the average of three replicates ± SD.

Expression of TERF1 in tobacco alters the expression ofsugar responsive genes

Because of the glucose response of TERF1-expressing to-bacco, we then further carried out the investigations on theregulation of TERF1 in the expression of sugar responsive

Figure 5. TERF1 activates the expression of the GUS gene controlled

by sugar-responsive element (SURE).

Agrobacteria harboring the reporter were infiltrated into the leaves of

4-week-old tobacco seedlings and incubated for 48 h. The data for

GUS activity are averages of triplicate samples and three independent

experiments, compared with the controls (the interaction of TERF1 with

Min (standardized to 100)). Min indicates the minimal TATA promoter

fused with GUS in pBI121; SURE/W box indicate that 4× cis-elements

SURE and W box are inserted upstream of Min as positive reporters

respectively; TERF1 as a positive effector indicates the full length of

TERF1 gene was driven by 35S promoter; “–” indicates no TERF1

effector plasmid but internal effector pBIN 35S mGFP4 was added.

genes. It has been shown that glucose and phytohormonesregulate the transcription of a variety of genes involved indifferent cellular processes, such as vegetative development,glucose metabolism and photosynthesis (Leon and Sheen 2003;Baena-Gonza′lez et al. 2007). The expression of several genesinvolved in photosynthesis is known to be regulated by sugars(Dai et al. 1999; Arenas-Huertero et al. 2000; Pego et al. 2000;Moore et al. 2003). In Arabidopsis, the effect of glucose onthe expression of the chlorophyll a/b-binding protein (CAB),ribulose-1,5-bisphosphate carboxylase small subunit (RBCS)gene was examined. The transcripts of these two importantphotosynthesis genes were significantly reduced in the pres-ence of glucose (Jang et al. 1997; Zhou et al. 1998). Since ourresults showed that OE seedling exhibited glucose sensitivity, itwas important to test whether these photosynthesis genes wereaffected by glucose. Our results indicated that the expressionlevels of NtCAB7, NtRBCS and NtHXK1a in OE seedlingwere much higher than those in WT (Figure 6). Especiallythe transcripts of CAB7 in OE lines were nearly sixfold higher


Figure 6. Expression of TERF1 in tobacco activates the expression of

sugar responsive genes.

Normally growing 4-week-old tobaccos were used for detecting the

expression of TERF1-downstream genes in tobacco. The expression

levels are relative to the control treatment of wild type (WT) that is taken

as 100 after normalizing against the actin signal, using cDNAs converted

from RNA samples of tobacco leaves as template of polymerase chain

reaction (PCR) amplifications. Error bars are based on three indepen-

dent experiments.

than that in the wild type, supporting the observation of darkgreen leaves in expression of TERF1 tobacco. Unlike the wildtype, the transcript levels of these genes were not obviouslydecreased after glucose treatment in OE seedlings (data notshown), indicating that expression of TERF1 in tobacco altersthe expression of sugar-related genes.

Discussion

Ethylene response factor proteins regulate multiple plant bioticand abiotic stress responses. Our previous report providedevidence that tomato ERF protein TERF1 is a member of theERF subfamily, and that expression of TERF1 in tobacco resultsin an increased level of endogenous ethylene and exhibitstypical phenotype of the ethylene response, and darker-greenleaves (Huang et al. 2004). Furthermore TERF1 confers hyper-sensitivity to ABA and tolerance to drought through activatingthe expression of osmotic stress-related genes with ethylene-dependent and -independent regulation in tobacco (Zhang et al.2005). In the present paper, we further establish that TERF1displays transcriptional regulation in glucose response, possiblythrough interaction with the sugar responsive element SURE.

As a major energy source and structural storage component,sugars act to influence plant development. Being sessile organ-isms, plants generate their own carbon through photosynthesis.In general, low sugar status enhances photosynthesis, nutrientmobilization and export, the abundance of sugars promotes

growth and carbohydrate storage (Rolland et al. 2006), whereasthere is increasing evidence that sugar plays important roles asa signaling molecule. Glucose, as one of the more importantsugars, plays vital roles in plant growth and development bothas an energy source and a signaling molecule, which is furthercontrolled by phytohormones (Leon and Sheen 2003). In thepresent paper, we have observed that low levels of glucosepromote seedling development, confirming the importance ofthe energy source of sugar. Higher glucose (6%) significantlyretarded the development, showing smaller leaves, reducedfresh weight and shorter roots in OE lines. This phenomenonwas even more serious under 7% glucose: some of the OEseeds did not germinate and the cotyledon developed a yellowcolor (data not shown). The effects of mannitol on developmentare different, indicating that TERF1 confers vital roles in thesugar response. However, it is very difficult from our presentdata to distinguish glucose as a signaling molecule or energysource, or both.

The complex signaling network reveals a crosstalk that linksthe sugar response to plant stress hormones (Leon and Sheen2003; Rolland et al. 2006). Genetic and molecular studies ofsugar-signaling mutants in Arabidopsis have uncovered the inte-gration between sugar and ethylene signaling (Zhou et al. 1998;Yanagisawa et al. 2003). The phenotypes of the glucose insen-sitive mutant gin1 acting downstream of the HXK-dependentsignaling pathway, showing the smaller, darker-green rosettesphenotypes, could be mimicked by ethylene treatment in thewild type. Moreover GIN1 acts downstream of ETR1 in theethylene signaling pathway (Zhou et al. 1998). This connectionis further supported by the discovery of ethylene overproduction(eto1) and ethylene constitutive signaling (ctr1/gin4) mutantsthat are glucose-insensitive (Zhou et al. 1998; Arroyo et al.2003), and ethylene-insensitive mutants (etr1, ein2, ein3 andein6) that display oversensitivity to glucose (Leon and Sheen2003). More interestingly, glucose antagonizes ethylene signal-ing by enhancing proteasome-dependent degradation of EIN3(Yanagisawa et al. 2003). In order to address the relationshipof TERF1-associated ethylene biosynthesis (Huang et al. 2004)and TERF1-enhanced sugar sensitivity, we further conductedthe assays using ethylene perception inhibitor AgNO3 duringroot development. Our preliminary results indicate that ethyleneaction and glucose response antagonistically affects root elon-gation (Huang et al. unpubl. data, 2008).

We noticed that the putative PERE appears within the pro-moter region of TERF1. PERE in the promoter of ArabidopsisERF1 is proven to be involved in the ethylene signaling pathway,which is the immediate downstream target of EIN3 (Solanoet al. 1998). Analysis with protein sequences indicates thatTERF1 shares 78% homology with ERF1. Further investigationswith yeast-one-hybrid and transient GUS expression providedevidence that LeEIL3 and LeEIL4, which share 63% and 68%similarity at amino acid levels with EIN3, respectively (Tiemanet al. 2001; Yokotani et al. 2003), interact with the promoter


of TERF1 (Huang et al. unpubl. data, 2008). Combining theobservation that OE seedlings display typical ethylene triple-response and enhance the expression of ethylene responsiveGCC box-containing genes (Huang et al. 2004), we proposethat TERF1 might function as a subset ethylene signaling asthe downstream of ethylene pathway component LeEIL3/4.

Sugar signaling can be divided into three steps: sugar sens-ing, signal transduction, and target gene expression. Unlike thehormone signal molecule, there are no common cis-elements asa marker for the sugar signaling pathway, which increases thedifficulty to target the downstream genes of sugar responsivetranscription factors. Emerging reports reveal that transcriptionfactors might be of relevance to plant sugar signaling, suchas SPF1, SUSIBA2 and STK (Ishiguro and Nakamura 1994;Zourelidou et al. 2002; Sun et al. 2003). Due to the unknowngenomic sequence of tobacco, there are limited data on tobaccogenes that contain target elements in their promoters; and it isdifficult to determine the sugar-regulated genes that contain cor-responding known cis-element. Thus, we chose several genesinvolved in photosynthesis to determine the influence of TERF1on gene expression. For instance, NtCAB genes encode chloro-phyll a/b binding proteins. CAB polypeptides bind to chlorophylla/b and carotenoid pigments, and then form a light-harvestingcomplex of photosystem II (PS II) in the thylakoid membranesof chloroplasts. This pigment/protein complex absorbs lightand transfers the resulting excitation energy to photo systemreaction centers (Castresana et al. 1988). Rubisco, encodedby the ribulose 1,5-bisphosphate carboxylase/oxygenase smallsubunit, is a key enzyme that fixes carbon dioxide to supportlife activity (Dhingra et al. 2004). The increased expression ofthe photosynthesis gene might result in the accumulation ofphotosynthesis products and sugar contents. The observationthat the expression of TERF1 increases the transcript levels ofphotosynthesis genes is consistent with the phenotype that ex-pressing TERF1 leads to dark green leaves in tobacco (Huanget al. 2004). To maintain the balance of energy and metabolism,the HXK levels in transgenic lines became higher, which mightenhance the sensitivity of sugar. Therefore, the elevated expres-sion of glucose-related genes in overexpressing TERF1 plantsis in agreement with a glucose-sensitive phenotype. Thus, wespeculate that expressing TERF1 in tobacco might affect theHXK-mediated signal transduction pathway, through interac-tion with SURE to activate the expression of sugar-relatedgenes.

In higher plants, sugars play vital roles in plant development.But sugar signaling transduction is confused, because of thedual function of sugars as nutrients and signaling molecules.This complexity is increased due to the source-sink interactionsand the cross-talk between the signaling network governed byplant hormones and environmental conditions. In Arabidopsis,two protein kinases, KIN10 and KIN11 have been proven toact as energy sensors and integrators that link stress, sugarand developmental signals (Baena-Gonza′lez et al. 2007). In

the present paper, our research establishes that ERF proteinTERF1 participates in the glucose response. Therefore, it willbe very interesting to analyze whether TERF1 is a commonintegrator in ethylene and sugar pathways.

Material and methods

Plant material and growth conditions

All plants were grown in growth chambers at 25 ◦C with a 16:8 hlight : dark cycle. For detecting the expression of TERF1, theleaves of 2-week-old tomato (Solanum lycopersicum (Lycoper-sicon esculentum) cv Lichun) plants were sprayed with glucoseor mannitol solution. For detecting the expression of TERF1-downstream genes in tobacco (Nicotiana tabacum cv NC89),leaves from normally growing 4-week-old plants were used. Thetransgenic tobacco plants expressing TERF1 were generatedas described by Huang et al. (2004). T3 tobacco plants wereused in this paper. Wild type and TERF1-expressing tobaccoare indicated as WT and OE, respectively.

TAIL-PCR assay

The genomic DNA of tomato Lichun was used as a template.TAIL-PCR (thermal asymmetric interlaced-polymerase chainreaction) was carried out as described by Liu et al. (1995). Thesequences of specific primers for TERF1 are as follows:

P1: 5′-CATCAATATCAACAAATGGATC-3′; P2: 5′-TCCATGATGATTCATCTTCC-3′ P3: 5′-AACTTCGTTACGGAATCTGTA-3′.

Germination, root and seedling development assays

All seeds in the following assays were first surface-sterilized,and kept at 4 ◦C for 3 d to break dormancy, and then the followingtreatments were carried out as described. For germinationassay, the tobacco seeds were kept in the growth chamberin MS medium containing glucose or mannitol. Germinationseeds were recorded every day by counting the proportion ofseeds with obvious protrusion of the radicle through the seedcoat.

For root elongation assay, the tobacco seeds were sown onMS basal salt supplemented with 1.0% agar in Petri dishes andplaced vertically in the growth chamber. After the roots wereabout 1 cm in length, the tobacco seedlings were transferred tothe same medium supplemented with glucose or mannitol, thetop of the root was marked and placed vertically in the growthchamber with its leaves in the upper space. After growth for7 d, root elongations were measured. Relative root elongationis referred to as the comparison with that of WT on MS withoutglucose.


For seedling development test, germinated seeds grown onMS basal salt plus 0.6% agar were transferred to the samemedium containing glucose or mannitol. The fresh weight ofsingle seedlings was measured at 10 d after treatment. Therelative fresh weight is referred to the comparison with that ofWT on MS without glucose.

β-glucuronidase transient assay in vivo

For construction of the reporter vectors, cis-acting regulatoryelements SURE (AAAACCGAA) or W box (TGACT) sequencesthat had been repeated four times were inserted into theupstream of the minimal TATA box (−46 to +10) to replacethe cauliflower mosaic virus (CaMV) 35S promoter in pBI121(Clontech, Palo Alto, CA, USA). For constructing the effectorvector, the β-glucuronidase (GUS) gene in pBI121 was replacedwith the full encoding region of TERF1, yielding the constructpTERF1. The plasmids of pTERF1 and pBIN 35S mGFP4GFP(as an internal control) were then separately introduced into theAgrobacterium tumefaciens strain LBA4404. Agrobacterium-mediated transient assay was carried out on the leaves of 4-week-old wild type plants as described by Yang et al. (2000).The GUS activity was measured for approximately 48 h incuba-tion at 25 ◦C with a 16:8 h light : dark cycle.

Transcriptional expression analyses

Reverse transcription-polymerase chain reaction experimentswere carried out using 1 μg of total RNA extracted byTrizol Reagent (Dingguo, Beijing, China). The first strandcDNA was synthesized with M-MLV reverse transcriptase(Promega, Madison, WI, USA) and oligo (dT) according to themanufacturer’s instructions. The following PCR amplificationswere carried out using the gene-specific primers. The specificprimers used and the corresponding size products andGene Bank accession numbers are given bellow: TERF1(5′-ATGTCAAGCCCACTAGAGAT-3′ and 5′-CTATGATGAAGTCATTAAAAGC-3′; 675 bp; AY044236); Tomato Actin Tom41(5′-CATGCCATTCTCCGTCTTGA-3′ and 5′-GCTAGGAGCCAATGCAGT-3′; 453 bp; U60480); Tobacco Actin (5′-CCACACAGGTGTGATGGTTG-3′ and 5′-CACGTCGCACTTCATGATCG-3′; 442 bp; X63603); tobacco CAB7 (5′-GTTACTTGACTGGTGAGTTC-3′and 5′-GGACAAAGTTTGTGGCATAG-3′; 588 bp; X58229); tobacco RBCS (5′-GAAGTACGAGACTCTCTCAT-3′ and 5′-CTGATGCACTGCACTTGAC-3′; 312 bp; X02353); tobacco HXK1a (5′-GCGTTGGATACTAATAGTTTG-3′ and 5′-GTACATTGAGTTAGAGGCAG-3′; 576 bp; AY553214).

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Supporting Information

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Figure S1. Sequence of the TERF1 promoter showing deducedcis-acting elements.

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Ethylene Response Factor TERF1 Enhances Glucose Sensitivity in Tobacco through Activating the Expression of Sugar-related Genes