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Plant & Cell Physiology 45(11): 1566-1577 (2004)

Local Induction of the alc Gene Switch in Transgenic Tobacco Plants by Acetaldehyde

Sara Schaarschmidt1, Nan Qu2,3, Dieter Strack1, Uwe Sonnewald2, Bettina Hause1

1Leibniz-Institut für Pflanzenbiochemie (IPB), Weinberg 3, D-06120 Halle (Saale), Germany 2Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany

eigener Anteil an der Publikation:

Planung und Durchführung der Versuche incl. - Anzucht, Pflege und Ernte der Pflanzen sowie - sämtlichen nachfolgenden Aufbereitungen und Analysen des Materials

ausgenommen einiger am IPK Gatersleben durchgeführten Zuckerbestimmungen (von ca. der Hälfte der in die Publikation eingegangenen Proben);

Auswertung der Versuche incl. Auswertung und Aufbereitung der Daten und Bilder; Verfassen der Publikation

jeweiliger Anteil der Co-Autoren:

Dr. Nan Qu

Transformation, Selektion und erste Charakterisierung der verwendeten Nicotiana tabacum alc::cwINV-Linien

Prof. Dr. Dieter Strack

teilweise Betreuung der Arbeit; Bereitstellung des Arbeitsplatzes; Korrekturlesen des Manuskripts

Prof. Dr. Uwe Sonnewald

Bereitstellung des Saatguts der verwendeten transgenen Pflanzen (Nicotiana tabacum alc::cwINV, NT alc::uidA); Diskussion der Daten; Korrekturlesen des Manuskripts

PD Dr. Dr. Bettina Hause

direkte Betreuung der Arbeit; Diskussion der Daten; Hilfe beim Verfassen der Publika-tion

Plant Cell Physiol. 45(11): 1566–1577 (2004)JSPP © 2004

1566

Local Induction of the alc Gene Switch in Transgenic Tobacco Plants by Acetaldehyde

Sara Schaarschmidt 1, Nan Qu 2, 3, Dieter Strack 1, Uwe Sonnewald 2 and Bettina Hause 1, 4

1 Leibniz-Institut für Pflanzenbiochemie (IPB), Weinberg 3, D-06120 Halle (Saale), Germany 2 Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany

;The alc promoter system, derived from the filamen-

tous fungi Aspergillus nidulans, allows chemically regu-lated gene expression in plants and thereby the study ofgene function as well as metabolic and developmental proc-esses. In addition to ethanol, this system can be activated byacetaldehyde, described as the physiological inducer in A.nidulans. Here, we show that in contrast to ethanol, acetal-dehyde allows tissue-specific activation of the alc promoterin transgenic tobacco plants. Soil drenching with aqueousacetaldehyde solutions at a concentration of 0.05% (v/v)resulted in the rapid and temporary induction of the alcgene expression system exclusively in roots. In addition, thesplit root system allows activation to be restricted to thetreated part of the root. The temporary activation of the alcsystem by soil drenching with acetaldehyde could be pro-longed over several weeks by subsequent applications atintervals of 7 d. This effect was demonstrated for the root-specific induction of a yeast-derived apoplast-located inver-tase under the control of the alcohol-inducible promotersystem. In leaves, which exhibit a lower responsiveness toacetaldehyde than roots, the alc system was induced in thedirectly treated tissue only. Thus, acetaldehyde can be usedas a local inducer of the alc gene expression system intobacco plants.

Keywords: Acetaldehyde — alc system — Apoplastic inver-tase — Chemically inducible gene expression — Nicotianatabacum — Root-specific induction.

Abbreviations: BTH, benzothiadiazole; CaMV, cauliflowermosaic virus; EcR, ecdysone receptor; GUS, β-glucuronidase;GAPDH, glyceraldehyde phosphate dehydrogenase; VP16, herpessimplex virus protein 16.

Introduction

Chemically inducible systems that activate or inactivategene expression represent powerful tools to study gene functionas well as biochemical, physiological and developmental proc-esses in plants. Several chemically inducible promoter systemshave been developed (for review, see Gatz and Lenk 1998, Zuo

and Chua 2000, Padidam 2003). They are mostly based onmechanisms of non-plant gene regulation responding to exter-nal signals that do not usually appear in higher plants. Themost prominent examples are the tetracycline-triggered pro-moter systems based on the bacterial tetracycline repressorTetR, and the glucocortinoid-receptor-based steroid-induciblesystem. The tetracycline-inducible system represents a de-repression system in which the repressor protein TetR blocksthe target promoter by binding to tet operator sites, introducedinto the respective promoter (Gatz and Quail 1988, Gatz et al.1992). The antibiotic tetracycline abolishes the binding of TetRto DNA, thereby relieving the repression. In the tetracycline-inactivation system the repressor TetR is fused to an acidic acti-vation sequence of herpes simplex virus protein 16 (VP16)(Gossen and Bujard 1992, Weinmann et al. 1994). In this case,the repressor is converted into an activator (tTA), which losesits DNA-binding ability upon tetracycline binding to the TetRmoiety. The steroid-inducible system makes use of the ligand-binding domain of a mammalian-derived glucocorticoid recep-tor. Glucocorticoid-dependent transcription is based on theinhibitory interaction between the heat shock protein HSP90and the steroid-binding domain of the receptor, thus inactivat-ing the receptor. In the presence of a steroid, the ligand associ-ates with the receptor leading to the release of HSP90 andactivating the receptor. The glucocorticoid receptor-bindingdomain has been fused to different plant transcription factors(Aoyama and Chua 1997, Simon et al. 1996). For a transcrip-tional control system that can be combined with any gene, thereceptor is fused to a protein complex consisting of the GAL4DNA-binding domain of yeast and the VP16 activation domain,forming all together the GVG activator (Aoyama and Chua1997). Both receptors can be combined to develop a geneexpression system inducible by the steroid dexamethasone andrepressible by tetracycline (Boehner et al. 1999). Inducible sys-tems that could be activated by non-steroidal agonists have alsobeen developed, based on the insect ecdysone receptor (EcR)and the use of registered agrochemicals as inducing agents(Martinez et al. 1999, Padidam et al. 2003). Furthermore, someendogenous plant promoters responding to specific chemicalshave been isolated, like the PR-1a promoter, which responds tobenzothiadiazole (BTH) (Goerlach et al. 1996).

For the widespread application of chemically induciblesystems in plants, specific characteristics of the inducers are

3 Present address: Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Strasse 8, D-07745 Jena, Germany4 Corresponding author: E-mail, [email protected]; Fax, +49-345-5582-1509.

Acetaldehyde as local inducer of alc gene switch 1567

required. Such features are in particular a high specificity forthe transgene and the compatibility of the plant and the envi-ronment. Furthermore, the inducers should allow convenientapplication with high efficiency at low concentrations, result-ing in low use rates and low costs.

Most endogenous plant promoters are not highly specificfor the transgene, leading to an additional induction of geneswhich are also controlled by the regulatory sequence. There-fore, the use of well-characterized regulatory elements fromevolutionarily distant organisms, like yeast or mammalians, isfavored. But, not all of the available non-plant gene regulationsystems really accomplish these requirements. For example,activation of the glucocorticoid-inducible transcription systemby dexamethasone can cause severe growth defects and theinduction of defense-related genes in Arabidopsis (Kang et al.1999). These effects were suggested to result from the systemitself, either by the nuclear localization of GVG after glucocor-ticoid binding or due to the potent VP16 transactivator domain.The tetracycline-inducible system requires high concentrationsof the repressor TetR due to the fact that the repressor competeswith endogenous transcription factors for access to the targetpromoter binding site. Even though the system could be suc-cessfully established for tobacco and potato, it does not work inArabidopsis, which possibly does not tolerate the amounts ofTetR needed for efficient repression. In some cases high levelsof the repressor were also shown to have adverse effects ontomato plants (Corlett et al. 1996).

A promising alternative to the systems mentioned abovehas been recently established (Salter et al. 1998). The alcohol-inducible alc gene expression system is based on the regula-tory elements of the strongly inducible Aspergillus nidulansalcA promoter. The transcriptional activation of the alcA geneby ethanol or other alcohols and small ketones like 2-butanone(Flipphi et al. 2002) is mediated by the AlcR transactivator, aDNA-binding protein belonging to the C6 zinc binuclear clus-ter family (Panozzo et al. 1997; for detailed review of the fun-gal system, see Felenbok et al. 2001). In plants, the regulatoralcR is expressed under the control of the cauliflower mosaicvirus (CaMV) 35S promoter, so that in the presence of a chemi-cal inducer AlcR will activate the expression of any gene fusedto a modified alcA promoter (Caddick et al. 1998, Salter et al.1998). The alcohol-inducible gene expression system couldalready be established for different plants like Arabidopsis thal-iana, Nicotiana tabacum, Solanum tuberosum and Brassicanapus using ethanol as inducer (Roslan et al. 2001, Salter et al.1998, Sweetman et al. 2002). Furthermore, recent studies indi-cate that the alc system can also be used for the temporal andspatial control of gene silencing by the inducible expression ofdouble-stranded RNA (Chen et al. 2003). Since acetaldehydewas proposed to be the physiological inducer of the alc genesystem (Flipphi et al. 2002), first investigations have been car-ried out testing this substance in transgenic systems. Data indi-cated a more rapid response of potato (S. tuberosum) plantscarrying the alc system to treatment with acetaldehyde com-

pared with ethanol (Junker et al. 2003). Moreover, a low effectof acetaldehyde on the plant metabolism could be shown. Toachieve tissue-specific induction of reporter gene expression,the alc promoter system was combined with tissue-specificpromoters to control the expression of alcR. In potato, the B33patatin promoter was used to induce the alc system and under-lying genes tuber specifically (Junker et al. 2003). A restrictedactivation of the alc system in Arabidopsis using different pro-moter combinations was shown by Deveaux et al. (2003). Theauthors were able to express the alcohol-regulated transcrip-tion factor AlcR limited to subdomains of the shoot apical andfloral meristems. This aspect of temporal as well as spatial con-trol of gene regulation provides a powerful tool for studyinggene function during plant development as well as physiologi-cal and metabolical processes in defined plant tissues.

To enhance the potential of tissue-specific activation ofgenes of interest by the alc system without the necessity of tis-sue-specific promoters, we investigated the effect of acetalde-hyde on the alc system in specific plant tissues of transgenictobacco (N. tabacum) plants. Using alc::uidA plants, we char-acterized the responsiveness of roots and single leaves toacetaldehyde by testing different concentrations as well asplant tissue of different ages. Furthermore, we optimized theacetaldehyde application to obtain long-lasting activation of thealc system in roots. Using tobacco plants expressing an apo-plast-located yeast-derived invertase under the control of thealcohol-inducible promoter, we demonstrated the functionalinduction of the enzyme upon drenching with acetaldehyde,leading to a root-restricted alteration of the ratio between hex-oses and sucrose.

Results

Root-specific induction by soil drenchingTo examine the potential applicability of ethanol and

acetaldehyde to a root-specific activation of the alc promotersystem, tobacco plants carrying the alc::uidA construct weresoil-drenched with different concentrations of each of theinducers. Ethanol concentrations in the range 0.05–0.5% (v/v)applied to the root system resulted in a relatively slight increasein β-glucuronidase (GUS) activity in roots (Fig. 1a). Despitethe bagging of pots immediately after treatment, concentra-tions of >0.2% (v/v) showed strong induction of the alc systemin young leaves, whereas GUS activity in roots showed a slightincrease only. In contrast, application of acetaldehyde at verylow concentrations resulted in marked root-specific inductionof the alc promoter system (Fig. 1b). In particular, acetalde-hyde concentrations of 0.01–0.1% (v/v) led to a nearly propor-tional increase in GUS activity in roots, whereas concentrationsup to 0.05% (v/v) had no detectable effect on GUS activity inleaves. Higher concentrations showed only a slight induction inleaf tissue. Drenching with acetaldehyde at concentrations upto 0.1% (v/v) seemed to have no negative effect on plant vigoras shown by the total protein content and activity of glyceralde-

Acetaldehyde as local inducer of alc gene switch1568

hyde phosphate dehydrogenase (GAPDH) as internal controlenzyme (Fig. 1c). The protein content of the roots remainedconstant upon treatment with acetaldehyde and the GAPDHactivity did not decrease until acetaldehyde concentrationsexceeded 0.1% (v/v). Acetaldehyde at a concentration of 0.2%(v/v) might damage the root as indicated by a decrease inGAPDH and GUS activity.

According to the presented results, further experiments onthe root-specific induction of the alc gene expression systemwere carried out using 0.05% (v/v) acetaldehyde. Soil drench-ing of plants in unbagged pots did not cause changes in theactivation levels in roots and leaves compared with plants inbagged pots (data not shown). Therefore, bagging of the potswas omitted in the following experiments. Flooding of theplants with water for 5 and 12 d, which possibly results inanaerobic conditions, did not affect the alc system in roots orleaves of transgenic tobacco plants (data not shown).

All acetaldehyde concentrations tested showed a transienteffect indicated by a decrease in GUS activity 8 d after applica-tion down to 10% or 20% of the activity shown 2 d after treat-ment (data not shown). To achieve long-lasting effects wetested repetitions of acetaldehyde application at different inter-vals. Alc::uidA tobacco plants were drenched with 0.05% (v/v)acetaldehyde once, twice (data not shown) or three times atintervals of 24 h and 48 h, respectively. Controls treated withwater showed no activation of the alc promoter (data notshown). Activation in leaves was not detectable in all experi-ments performed using acetaldehyde. Fig. 2 shows the induc-tion of GUS activity in roots. A single drenching withacetaldehyde resulted in an increase in GUS activity lastingabout 1 week (Fig. 2a). The GAPDH activity of roots remainedat a nearly constant level. Repeated acetaldehyde application atintervals of 24 h did not significantly increase GUS activity(Fig. 2b). In addition, decreasing GAPDH activity indicated anegative effect on plant vigor of successive treatments at shortintervals. Triple drenching at intervals of 48 h resulted in anearly linear increase in GUS activity (Fig. 2c). After a slightdecrease, GAPDH activity increased to the previous level indi-cating the plant’s ability for rapid recovery. However, a long-lasting effect on the induction of GUS activity could not beobtained. GUS activity decreased again rapidly 9 d after thefirst treatment. Thus, to activate the alc system over a longperiod subsequent applications of acetaldehyde at greater timeintervals seemed to be more appropriate as shown for the long-lasting induction of a yeast-derived invertase by weekly acetal-dehyde treatment (see below).

To analyze a possible effect of plant development on theactivation of the alcohol-inducible promoter system in roots,alc::uidA tobacco plants of various ages were soil-drenchedwith acetaldehyde (Fig. 2d). By this, it could be shown that theresponse to acetaldehyde was age dependent. In particular, inroots of 6- to 8-week-old plants soil drenching with 0.05% (v/v) acetaldehyde caused 2- to 5-fold higher GUS activity than inroots of 10- to 14-week-old plants, indicating higher respon-siveness of young plants.

To visualize the root-specific induction of the alc pro-moter by drenching with acetaldehyde, histological GUS deter-mination was carried out. In accordance with the presentedresults based on the fluorimetric determination of GUS activ-ity, plants treated once with 0.05% (v/v) acetaldehyde exhib-ited root-located GUS staining (Fig. 3a). Roots showed a

Fig. 1 Activation of the alc system by different concentrations of eth-anol and acetaldehyde, respectively. GUS activity in roots (squares),young leaves (triangles) and older leaves (circles) of 8-week-old alc::uidA tobacco plants 2 d after soil drenching once with 100 ml dilutedethanol (a) or acetaldehyde (b). To prevent formation of vapor, potswere bagged immediately after treatment. After soil drenching, etha-nol- and acetaldehyde-treated plants were cultivated in separate green-house chambers under similar growth conditions. (c) GAPDH activityand protein content of roots treated with different acetaldehyde con-centrations (n = 3).


stronger staining 2 d after application of acetaldehyde com-pared with 6 d after treatment. Moreover, root tips were stainedonly at 2 d but not at 6 d after treatment. This suggested transientinduction of the alc system by acetaldehyde, because new-growntissue was not affected by previous acetaldehyde application.

To test the possible translocation of the inducer, root sys-tems of alc::uidA tobacco plants were split onto two pots. Onepart of the root system was triple treated with 0.05% acetalde-hyde at intervals of 48 h, whereas the other part was treatedwith water (Fig. 3b). Both histological and fluorimetrical deter-mination of GUS activity demonstrated induction restricted tothe acetaldehyde-treated part of the root system. In both partsGAPDH activity was almost unaffected. Therefore, drenchingwith acetaldehyde at a concentration of 0.05% (v/v) activatedthe alc system only in roots treated directly with acetaldehyde.This allows root-specific induction of genes of interest underthe control of the alc promoter in transgenic plants as shownbelow for alc::cwINV tobacco plants.

Leaf-specific induction by infiltrationTo test whether acetaldehyde can also be used for local

gene activation in above-ground plant tissues, leaves of alc::uidtobacco plants were treated with different concentrations ofacetaldehyde by infiltration into the intercostal regions (Fig.4a). Histochemical GUS determinations revealed lower respon-siveness of leaves to acetaldehyde as compared with roots. Inparticular, treatment of medium-aged leaves with acetaldehydeconcentrations up to 1% (v/v) resulted in no GUS staining,whereas infiltration with concentrations of 2% (v/v) and 5% (v/v) led to an induction of the GUS activity in and directlyaround the treated leaf area. The same effect could be obtainedfor acetaldehyde application by dipping and brushing (data notshown). Fluorimetric GUS determination indicated that theinduction of the alc system by infiltration of 5% (v/v) acetalde-hyde is highly specific for the treated leaf (Fig. 4b). Thedirectly treated leaf area showed the highest induction of GUSactivity. Less activity could be detected in the surrounding tis-sue as shown before by histological GUS staining. Other planttissues, like the upper and lower leaves, stem and leaf stalks aswell as roots, exhibited negligible GUS activity (Fig. 4b). Fur-thermore, the local effect of acetaldehyde in leaves was corrob-

orated by the histological GUS analysis of leaves showing nostaining of leaf veins (Fig. 4a, insets).

To analyze the response of leaves at different developmen-tal stages to acetaldehyde, histochemical and fluorimetric GUS

Fig. 2 Time courses of the induction of GUS activity by repeated soildrenching with acetaldehyde. (a) GUS activity in roots (squares),young leaves (triangles) and older leaves (circles) of 8-week-old alc::uidA tobacco plants after a single treatment with 100 ml 0.05% (v/v)acetaldehyde. Soil drenching with acetaldehyde was repeated at inter-vals of 24 h (b) and 48 h (c). Arrows indicate time points of applica-tion. As internal control for plant vitality the GAPDH activity of rootswas determined (cross). (n = 3). (d) Influence of plant age on theinduction of the alc gene expression system by soil drenching withacetaldehyde. alc::uidA tobacco plants of different ages were drenchedtwice, each time with 100 ml 0.05% (v/v) acetaldehyde with an inter-val of 48 h. GUS activity was determined 4 d after the first treatment(n = 6).


determinations were carried out using leaves from transgenictobacco plants of differing ages injected with 5% acetaldehyde(Fig. 4c, d). According to the results from soil drenching of dif-ferent-aged plants, treated leaf areas of the youngest leavesshowed the highest responsiveness for acetaldehyde. Withincreasing leaf age, protein content (data not shown) andGAPDH activity (Fig. 4d) decreased, indicating a generallydecreasing activity of older leaves. Nevertheless, infiltration of5% (v/v) acetaldehyde into the leaf did not affect the GAPDHactivity in leaves or other plant tissue (Fig. 4b, d). In conclu-sion, acetaldehyde can be used as a local inducer of the alcgene expression system in leaves of transgenic tobacco plants,albeit at high concentrations.

Root-specific induction of a yeast-derived apoplastic invertaseunder the control of the alc promoter

As described above, the alc system and its physiologicalinducer acetaldehyde provide a time-controlled and locallyrestricted induction of genes of interest in transgenic plants. Todemonstrate metabolic changes as a result of this induction, weused transgenic tobacco plants harboring the alc::cwINV con-struct. These plants carry a chimeric yeast-derived invertasegene under the control of the alcohol-inducible promoter.Fused to the potato proteinase inhibitor II signal peptide, theinvertase is targeted to the apoplast. Homozygous plants of theT3 generation of three independent transgenic lines were testedand did not exhibit differences in induced invertase activitiesupon soil drenching with acetaldehyde (data not shown).

Fig. 3 Localization of GUS activity in roots induced by soil drenching with acetaldehyde. (a) GUS staining of 6-week-old alc::uidA tobaccoplants drenched once with 100 ml 0.05% (v/v) acetaldehyde. Whole plants were fixed and stained 2 and 6 d after treatment, respectively. Insetsshow a representative root tip. Bars represent 1 cm and 50 µm in the case of insets. (b) GUS staining and determination of GUS activity of water-and acetaldehyde-treated root parts of one representative 12-week-old alc::uidA tobacco plant. The alc system was induced by triple soil drench-ing with 100 ml each of 0.05% (v/v) acetaldehyde at time intervals of 48 h. Seven days after the initial application a small amount of root mate-rial was taken from each root part for fluorimetric GUS determination (small columns). The remaining material was immediately fixed and usedfor GUS staining. As internal control for the vitality of the root parts GAPDH activity was measured (wide columns). H2O, water-treated rootpart; Aa, acetaldehye-treated root part of one and the same plant. Bar represents 1 cm.

Fig. 4 Activation of the alc system in leaves of alc::uidA tobacco plants by acetaldehyde. (a) GUS staining of medium-aged leaves of 8-week-old plants injected with acetaldehyde at different concentrations. Treated leaf areas were marked directly after application. Leaves were fixed andstained 2 d after treatment. Insets show stained leaf areas after injection of 2% and 5% acetaldehyde, respectively, into the intercostal regions.Bars represent 1 cm. (b) GUS and GAPDH activities in different plant tissues 2 d after leaf-treatment using acetaldehyde (open columns) in com-parison with untreated plants (filled columns). In 8-week-old alc::uidA tobacco plants two intercostal regions of a medium-aged leaf were injectedwith 5% (v/v) acetaldehyde. Fluorimetric GUS determination and determination of GAPDH activity as internal control for the vitality of theplants were carried out for the directly treated leaf area, the surrounding leaf tissue, upper leaves, lower leaves, stems and leaf stalks as well as forroots. Values represent the mean of three plants. (c) Histochemical GUS analysis of leaves of different developmental stages. GUS staining wascarried out 2 d after infiltration of two intercostal regions of each leaf with 5% (v/v) acetaldehyde. Treated leaf areas were marked immediatelyafter infiltration. The whole plant was fixed and stained. Insets show stained leaf areas of leaf numbers 1 and 3. Leaf number 1 presents theyoungest, leaf number 7 the oldest fully developed leaf of each plant. Bar represents 1 cm. (d) Determination of GUS and GAPDH activities inleaves of different developmental stages. One intercostal region of each leaf of 8-week-old alc::uidA tobacco plants was injected with 5% (v/v)acetaldehyde. The enzyme activities were measured 2 d after treatment and are shown for the directly treated leaf areas (filled columns) and thesurrounding untreated leaf areas (open columns). (n = 3).


Fig. 4 (a–d)


Therefore, these lines were not separated in the followingexperiments, giving mean values of all three lines.

Invertase in situ staining visualized the invertase activityinduced by soil drenching with acetaldehyde (Fig. 5). Water-treated transgenic plants showed only faint staining of the rootsystem, whereas plants drenched once with 0.05% acetalde-hyde exhibited strong staining of the entire root system (Fig.5a, c). The negative control, performed by omitting sucrose inthe staining solution, showed no background staining due toremaining glucose in root tissue (Fig. 5b). Staining of cross-sections of roots indicated a high invertase activity in all rootcells after induction of the alc system by acetaldehyde (Fig. 5f,g). Thus, the induction was not limited to specific cell types.All controls performed with sections showed no staining (Fig.5d, e).

To achieve long-lasting induction of the alc system,drenching with 100 ml 0.05% (v/v) acetaldehyde three timesonce per week has been carried out (Fig. 6). By these treat-ments, the invertase activity could be induced in roots over atleast 4 weeks (Fig. 6a). Neither young nor old leaves showedincreased invertase activity induced by acetaldehyde (Fig. 6b).

Variations in the supply of light and nutrients, causing severephenotypic effects, had no significant effect on the invertaseactivity (data not shown). Increasing invertase activity resultedin an accumulation of glucose and fructose and decreasingamounts of sucrose in roots. The time course of the ratiobetween the amounts of these two hexoses and sucrose corre-lated with the detected invertase activity (Fig. 6c). Thus, theyeast-derived extracellular invertase was shown to be function-ally induced upon acetaldehyde treatment in alc::cwINVtobacco plants.

Discussion

In this study, we established a system for the tissue-spe-cific activation of the alc gene switch, particularly in view of amodulation of root metabolism independently of the metabo-lism in above-ground tissues. We could demonstrate that etha-nol is not suitable for the exclusive activation of the alc systemin roots of transgenic tobacco plants (Fig. 1). This is in agree-ment with the fact that soil drenching with ethanol led to aninduction of the alcohol-inducible promoter in leaves already at

Fig. 5 In situ invertase staining of alc::cwINV tobacco roots drenched with acetaldehyde. For the localization of invertase activity 2 d afterinduction, 8-week-old alc::cwINV tobacco plants were soil-drenched once with 100 ml 0.05% (v/v). Control plants were water-treated. (a–c)Staining of whole root systems (30 min, 30°C). (d–e) Staining of root cross-sections of 200 µm thickness (48 h, 30°C). Incubation with stainingsolution containing 1% sucrose was carried out using water-treated (a, d) and acetaldehyde-treated (c, f, g) roots. As negative control, one part ofthe acetaldehyde-treated root system and cross-sections of these roots were incubated with staining solution without sucrose (b, e). Bars represent25 µm.


low concentrations (Salter et al. 1998). When applied by rootdrenching, the effect of ethanol on the alc promoter system inleaves was much stronger than after the direct treatment ofleaves by spraying. Moreover, it could be shown that ethanolvapor is an efficient inducer of the alc system in Arabidopsis(Roslan et al. 2001) as well as in tobacco, potato and oilseedrape (Sweetman et al. 2002). Ethanol vapor led to higher acti-vation levels in leaves than root drenching with ethanol. There-fore, we tried to circumvent the rise of ethanol vapor by

bagging the pots. This, however, could not prevent activation ofthe alc promoter in leaves. Another inducer of the alc pro-moter is acetaldehyde, which has recently been shown to be thesole physiological inducer of alc gene expression in A. nidu-lans (Flipphi et al. 2002). In transgenic potato plants, acetalde-hyde activated the alc system more rapidly and caused lessmetabolite changes than ethanol (Junker et al. 2003). Hence,we tested acetaldehyde for induction of the alc system intobacco to modulate plant metabolism by the expression ofunderlying genes. We demonstrated that soil drenching withacetaldehyde leads to root-specific activation of the alc geneexpression system in tobacco. The time course of GUS activityand GUS staining of root tips indicated a short-term effect ofacetaldehyde on the alc system (Fig. 2, 3). Moreover, weshowed that acetaldehyde allows restricted induction of the alcsystem in roots and root parts by soil drenching. Additionally,leaves in direct contact with acetaldehyde applied either byinfiltration, brushing or dipping showed an induction of the alcsystem restricted to the treated leaf area. Rising acetaldehydevapor did not seem to have any effect on alc gene expression.Histological and fluorimetrical GUS determinations indicatedthat acetaldehyde is not subsequently translocated in the planttissue and has no systemic effect in tobacco plants (Fig. 3, 4).For example, tobacco plants grown in the split root systemexhibited GUS activity only in the acetaldehyde-treated rootpart but not in the water-treated part. Infiltration of the intercos-tal regions of leaves with acetaldehyde did not result in theinduction of GUS activity in other plant tissue, particularly leafveins, stems and leaf stalks. This argues against the systemictransport of the inducer. In some cases, GUS activity could alsobe detected directly around the injected leaf area marked imme-diately after treatment. This effect seems to be due to restrictedspreading of acetaldehyde by diffusion in the intercostalregions without subsequent uptake into the veins. Neverthe-less, infiltration of acetaldehyde had no influence on the alcsystem in other plant tissues except of the treated leaf. Thistemporary and local effect of acetaldehyde permits activationof genes of interest by the alcohol-inducible promoter atdefined developmental stages in root parts as well as singleleaves and allows a direct comparison of induced and non-induced parts of the same plant. Another strategy for control-led tissue-restricted gene regulation has been recently shown(Deveaux et al. 2003, Junker et al. 2003, Maizel and Weigel2004). In these studies tissue-specific promoters were com-bined with the alc promoter system, allowing time controlledand highly tissue-specific activation of the alc system andunderlying genes. For tuber-specific induction in potato theB33 patatin promoter was used (Junker et al. 2003). Combina-tion of the alc system with the promoter of the meristem iden-tity gene LEAFY allowed flower-specific induction (Maizel andWeigel 2004). Deveaux et al. (2003) combined the alc systemwith regulatory sequences driving the alcR transcription factorin meristematic subdomains of Arabidopsis plants. Thus, pro-moter combinations were revealed as an elegant method, exhib-

Fig. 6 Time course of invertase activity and resulting effects on sugarcontents in alc::cwINV plants. To induce the yeast-derived invertase,8-week-old alc::cwINV tobacco plants were triple drenched with100 ml each of 0.05% (v/v) acetaldehyde at intervals of 7 d (filled tri-angles). As controls water-treated transgenic plants (open triangles)and acetaldehyde-treated wild-type plants (circles) were used. (a) Spe-cific invertase activity in roots (mean values of two independent exper-iments, n = 6). (b) Specific invertase activity in leaves (mean values ofyoung and older leaves, n = 6). (c) Ratio of the sum of the amounts ofglucose and fructose to the amount of sucrose in roots (mean values oftwo independent experiments, n = 6). Arrows indicate the time pointsof acetaldehyde application.


iting high specificity for restricted gene regulation even fortissue subdomains. However, this strategy requires specificconstructs containing the respective tissue-specific promoters.Irrespective of the work, those promoters are not yet availablefor every plant and every special tissue. Moreover, it could beshown by Junker et al. (2003) that using the alc system and theB33 patatin promoter the reporter gene expression was not uni-form but limited to the outer part of potato tubers. To date it isnot clear whether this effect is based on the activity of the pata-tin promoter or on the accessibility of cells to the inducingagent. Hence, constitutive expression of alcR under the CaMV35S promoter and the subsequent specific activation of the alcsystem and underlying genes by acetaldehyde offers a con-venient alternative for temporally and locally controlled generegulation.

Furthermore, we characterized the responsiveness of dif-ferent plant tissues for an application with acetaldehyde fordependence on the developmental stage. Roots showed a nearly100-fold higher response to acetaldehyde in comparison withleaves. This might be due to more efficient uptake of aqueousacetaldehyde solutions by roots than by leaves. Such an effecthas already been shown for the use of aqueous ethanol solu-tions as inducing agent (Salter et al. 1998). Here, soil drench-ing with 1% (v/v) ethanol revealed a higher effect on the alcsystem in leaves than direct leaf treatment by spraying with10% (v/v) ethanol. Additionally, rising ethanol vapor, probablyeasily taken up by aerial parts of the plant, was shown to be ahighly efficient inducer (Roslan et al. 2001, Sweetman et al.2002). Compared with ethanol, the wide differences in theresponsiveness of roots and leaves to acetaldehyde could bedue to the lack of effect of acetaldehyde vapor. Nevertheless, inboth tissues, the effect of acetaldehyde treatment decreased sig-nificantly with increasing plant or leaf age. Additionally, histo-chemical GUS determinations indicated the strongest GUSactivity upon soil drenching in young roots and particularly inroot tips. Thus, the decreasing effect of repeated soil drenchingwith acetaldehyde at intervals of 7 d, which has been shown forthe induction of a yeast-derived invertase in transgenic tobaccoplants (Fig. 6), could be due to the altered responsiveness of theplants. Adaptation of the plant to acetaldehyde, however, can-not be excluded, but it seems to be more likely that the decreas-ing effect of soil drenching is caused by the decreasingresponsiveness of plants over the weeks. The altered suscepti-bility of tissues of different developmental stages could be dueto the generally lower activity of older plant tissue. This couldaffect directly the responsiveness to the inducing agent ormight also affect the activity of the CaMV 35S promoterexpressing the alcR regulator. Previous studies have alreadyindicated that the CaMV promoter does not necessarily showthe same expression level in all tobacco tissues (Mitsuhara etal. 1996, Harper and Stewart 2000). In these studies the CaMVpromoter has been shown to be more active in young leavesthan in older ones. It has been suggested that the CaMV 35Spromoter exhibits higher activity in the S phase of the cell cycle

(Nagata et al. 1987). Furthermore, the CaMV promoter hasbeen shown to be highly active in roots, especially in root tips(Benfey et al. 1989). This could also explain the higher respon-siveness of roots in comparison with leaves.

Nevertheless, soil drenching with acetaldehyde three timesonce a week allows long-lasting induction of the alc systemover at least 1 month. In contrast, successive treatments at shortintervals as well as higher concentrations of acetaldehydemight affect plant vigor leading to lower activation levels (Fig.1, 2). This was for example demonstrated by tobacco rootstreated with acetaldehyde concentrations of >0.1% exhibitinglow GUS activity. This decreasing effect of higher acetalde-hyde concentrations has also been described for transgenicpotato tubers, but with 10-fold higher concentrations (Junker etal. 2003), again suggesting a plant- and tissue-specific optimalconcentration of the inducer. Moreover, as shown by Junker etal. (2003) acetaldehyde had low effects on the plant metabo-lism in an optimal range, which is specific for the respectiveplant system. Therefore, optimization of acetaldehyde applica-tion in view of the specific requirements has to be carried outas in any other chemically inducible system.

The presented data clearly demonstrate the opportunity foran easy to handle and temporally regulated, tissue-specific acti-vation of the alc promoter system in plants using acetaldehyde.To demonstrate the physiological significance of the system,we used alc::cwINV tobacco plants expressing a yeast-derivedinvertase under the control of the alcohol-inducible promoter.Constitutive induction of yeast-derived invertase in transgenictobacco plants resulted in dramatic changes in plant develop-ment and phenotype (von Schaewen et al. 1990, Sonnewald etal. 1991). Upon constitutive invertase expression plants showedstunted growth, bleached and necrotic lesions of leaves, areduced rate of photosynthesis and suppressed root formation.Furthermore, transgenic plants expressing the yeast-derived,apoplast-located invertase constitutively exhibited a higherdefense status due to increasing levels of phenylpropanoids(Baumert et al. 2001) as well as enhanced expression of PRprotein genes, increasing callose content, stimulated peroxi-dase activity and elevated salicylic acid levels (Herbers et al.1996). Using transgenic plants expressing the invertase underthe control of the alcohol-inducible promoter system, we couldinduce the apoplast-located invertase by drenching with acetal-dehyde root-specifically without a direct effect on the metabo-lism in leaves. Thus, the amounts of glucose, fructose andsucrose in leaves remained constant whereas acetaldehyde-treated roots showed an increasing amount of hexoses and adecreasing sucrose content compared with untreated roots. Wecould thus establish a powerful tool for studying metabolicprocesses like carbohydrate metabolism by modulation of rootmetabolism independently of the metabolism in above-groundtissue. This system provides the possibility to analyze the influ-ence of modulated carbohydrate metabolism on interactionsbetween plants and root-specific microorganisms, includingpathogens and symbionts.


Under optimized conditions, activation of the alc geneexpression system by acetaldehyde represents a highly compat-ible chemically regulated system and complements the possi-ble field of application given by ethanol, a well-characterizedinducer of the alc system. Summarizing the presented and pre-vious results (see Introduction), both acetaldehyde and ethanolprovide easy to handle, non-toxic and cheap activation of genesunder the control of the alcohol-inducible promoter showing ahigh specificity for the transgene. Both inducers exhibit highefficiency at low concentrations and complement one anotherto a wide range of usage including tissue-restricted induction aswell as widespread induction by either soil drenching, spray-ing, infiltration, brushing, dipping or vaporization. Conclud-ing, the alc gene expression system represents a chemicallyinducible system which can be activated in a multifaceted man-ner by the use of different inducers. In addition, time- and spa-tially controlled gene regulation by acetaldehyde will allow theinduction or silencing of genes, whose constitutive up- ordown-regulation would have deleterious effects on plant devel-opment or plant vigor.

Material and Methods

Material, growth conditionsWild-type tobacco plants (N. tabacum cv. Samsun NN) were

obtained from Vereinigte Saatzuchten eG (Ebsdorf, Germany). Seedsof alc::uidA tobacco plants were kindly provided by Ian Jepson (Syn-genta). All plants were germinated on solid MS medium (Duchefa,Haarlem, Netherlands). Transgenic plants were selected on mediumcontaining 50 mg litre–1 kanamycin A (Duchefa). After 3 weeks plantswere transferred into 12-cm pots filled with expanded clay (OriginalLamstedt Ton; Fibo ExClay, Lamstedt, Germany) and grown in thegreenhouse at 16 h light (22°C) and 8 h dark (20°C). Plants werewatered with distilled water and fertilized weekly with 5 ml Long Ash-ton (20% phosphate) (Hewitt 1966). Unless indicated otherwise,experiments were carried out with 8-week-old plants. For the extrac-tion of proteins, soluble sugars and starch, root and leaves were har-vested 3 h after beginning of the light period and rapidly frozen inliquid nitrogen. Data were obtained from three individual plants ofeach genotype and treatment. Each leaf sample for one developmentalstage of leaves was pooled from two leaves of the same plant.

Plasmid construction and plant transformationTo obtain inducible expression of apoplastic, yeast-derived inver-

tase, a chimeric gene comprising the proteinase inhibitor II signal pep-tide and the mature yeast invertase protein was PCR amplified fromplasmid PI-3-INV (von Schaewen et al. 1990). The PCR product wassubcloned into pGEM-T and subjected to sequence analysis. Thereaf-ter, the chimeric coding region was placed under the control of thealcohol-inducible expression system by inserting the fragment into theappropriate plant transformation vector as described in Caddick et al.(1998) yielding plasmid alc::cwINV. Subsequently, plasmid alc::cwINV was transformed into Agrobacterium tumefaciens, strainCV58C1, carrying the virulence plasmid pGV2260 (Deblaere et al.1985). Transformation of tobacco plants using Agrobacterium-medi-ated gene transfer was carried out as described previously (Rosahl etal. 1987).

Treatment with acetaldehyde and ethanolTo compare the effect of ethanol and acetaldehyde on the root-

specific activation of the alc system, tobacco plants were soil-drenchedonce with 100 ml of different concentrations of the two inducers.Afterwards, the pots were immediately enclosed in a plastic bag. Insubsequent soil-drenching experiments carried out with 0.05% (v/v)acetaldehyde, plant pots were placed in sealed plastic pots. To inducethe alc promoter in leaves, aqueous solutions of different acetaldehydeconcentrations were injected directly into the leaf. Treated areas weremarked immediately after infiltration.

Protein extraction and determination of enzyme activitiesFrozen plant material (80 ± 2.5 mg) was homogenized in liquid

nitrogen and incubated for 10 min on ice with 600 µl extraction buffercontaining 50 mM sodium phosphate (pH 7.0), 10 mM EDTA, 10 mM2-mercaptoethanol, 0.1% (w/v) N-lauroyl-sarcosine sodium salt, and0.1% (v/v) Triton X-100. After centrifugation (14,000×g, 10 min, 4°C)the supernatant was used for the determination of enzyme activities.All measurements were carried out in three independent replicates foreach sample. Protein content was determined as described by Brad-ford (1976) using bovine serum albumin (Roth, Karlsruhe, Germany)as the standard.

Protein extracts of root and leaf samples were assayed for GUSactivity by fluorometry (Jefferson et al. 1987). Measurement ofGAPDH activity was carried out by incubating 5 µl protein extractwith 200 µl reaction buffer containing 100 mM Tris–HCl (pH 7.0),5 mM MgCl2, 1 mM sodium fluoride, 5 mM DTT, 0.5 mM NADH,10 mM 3-phosphoglycerate, 10 U triose phosphate isomerase (Roche,Mannheim, Germany) and 20 U phosphoglycerate kinase (Sigma-Aldrich, Steinheim, Germany). The absorbance at 340 nm was meas-ured as kinetics using a 96-well microtitre plate reader (Sunrise;Tecan, Crailsheim, Germany). Boiled protein extract of each samplewas incubated and measured in the same manner as controls. Valueswere standardized using purified GAPDH (Roche). The activity of theyeast-derived invertase was determined in the neutral pH range to limitthe influence of endogenous plant invertases. For this, 5 µl extractwere incubated with 200 µl reaction buffer containing 100 mM imida-zole (pH 6.9), 200 mM sucrose, 10 mM MgCl2, 2 mM NAD, 1 mMATP, 0.5 U glucose-6-phosphatase dehydrogenase from Leuconostocmesenteroides (Serva, Heidelberg, Germany) and 1 U hexokinase(Fluka, Buchs, Switzerland). The increase in absorbance at 340 nmwas measured in a 96-well microtitre plate reader at intervals of10 min. Boiled protein extracts were used as controls. Assays werestandardized using purified yeast invertase (Fluka).

In situ stainingsLocalization of the GUS protein was carried out by staining as

described by Blume and Grierson (1997). After fixation, roots, leavesand whole plants were incubated with staining solution for 36 h at37°C. Chlorophyll was removed by washing in 90% (v/v) ethanol.

To localize the induction of invertase activity in acetaldehyde-treated alc::cwINV tobacco plants, root pieces of 3 mm were fixed in2% (w/v) paraformaldehyde, 2% (w/v) polyvinylpyrrolidone 40 and1 mM DTT in PBS (pH 7.0) at RT for 1 h (Sergeeva and Vreugdenhil2002). After washing in PBS, root pieces were immobilized in 15%(w/v) gelatine and sectioned into 200 µm thick cross-sections using avibrating blade microtome (VT 1000 S; Leica, Nussloch, Germany).Sections were collected in 10 mm tissue culture inserts with a 8 µMpolycarbonate membrane (Nunc A/S, Roskilde, Denmark) placed in12-well macrotitre plates. To remove soluble sugars sections werewashed with PBS at 4°C over night and afterwards stained for inver-tase activity using 1 ml staining buffer consisting of 38 mM sodiumphosphate (pH 6.0), 25 U glucose oxidase (Fluka), 0.024% (w/v)


nitroblue tetrazolium, 0.014% (w/v) phenazine methosulfate and 1%(w/v) sucrose (Doehlert and Felker 1987, Sergeeva and Vreugdenhil2002) for 48 h at 30°C in the dark. In control reactions sucrose wasomitted. Staining of whole root systems for 30 min at 30°C was car-ried out in the same way except for a more intensive washing beforestaining.

Determination of soluble sugarsFrozen root and leaf material (each sample: 50 mg FW) was

homogenized in liquid nitrogen and incubated with 500 µl 80% (v/v)aqueous ethanol in a thermomixer for 1 h at 80°C and 1,000 rpm. Aftercentrifugation (14,000×g, 5 min, 4°C) the supernatant was evaporatedto dryness at 20°C. Resuspended residues (in 250 µl aqua dest.) wereused for the determination of soluble sugars in three replicas. For this,200 µl buffer containing 50 mM imidazol (pH 6.9), 5 mM MgCl2,2 mM NAD, 1 mM ATP and 0.1 U glucose-6-phosphatase dehydroge-nase from L. mesenteroides were added to 10 µl of the extract. Theabsorbance at 340 nm was measured after incubation for 10 min at RTin a microplate reader (Sunrise). For the determination of glucose0.1 U hexokinase (Fluka) in 5 µl aqua dest. was added. The absorb-ance at 340 nm was measured when the reaction reached a plateau.The content of fructose and sucrose was quantified in the same wayusing 0.1 U phosphoglucose isomerase (Sigma-Aldrich) and 0.5 Uinvertase (Fluka), respectively, in 5 µl aqua dest. Values were calcu-lated from the differences between the measurements using glucoseand sucrose as standards.

Acknowledgments

We would like to thank Mohammad-Reza Hajirezaei for provid-ing the method for the determination of soluble sugars and for helpwith the measurement of sugar content. We are also grateful to Ian Jep-son for providing seeds of alc::uidA tobacco plants.

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(Received May 18, 2004; Accepted August 12, 2004)

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