primary metabolism in plant defense' · sis in nadp-me-type c, plants such as maize, sugar...

Plant Physiol. (1995) 108: 949-960

Primary Metabolism in Plant Defense'

Regulation of a Bean Malic Enzyme Cene Promoter in Transgenic Tobacco by Developmental and Environmental Cues

Jorg Schaaf, Michael Herbert Walter*, and Dieter Hess

Universitat Hohenheim, lnstitut f ü r Pflanzenphysiologie (260), D-70593 Stuttgart, Cermany

NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) catalyzes the oxidative decarboxylation of malate to pyruvate, producing CO, and NADPH. We have examined regulatory properties of a 2.8-kb promoter-leader fragment of a bean (Pbaseolus vurgaris 1.) NADP-ME gene (PvME1) predicted to encode a cytosolic form of the enzyme by expression analysis of promoter-P-glucuronidase fusions in transgenic tobacco plants. l h e PvMEl promoter directed strong expression in stems, which was confined to vascular and pith tissues, and was also active in floral and reproductive tissues. Wound- ing caused a marked induction of promoter activity, which was further strongly enhanced upon application of stimuli related to pathogen defense. Clutathione (reduced form) was the strongest inducer, but oxidized glutathione, funga1 elicitor, cellulase, catalase, ascorbic acid, and NADPH were additional potent promoter- stimulating agents. Responsiveness to reduced glutathione was also shown at the leve1 of PvMEl mRNA accumulation in bean plants. l h e putative contributions of NADP-ME gene expression to the plant defense response and possible mechanisms of defense gene regulation by conditions of oxidative stress as well as by H,O, and antioxidant levels are discussed.

The developmental program of metabolic activities in plants is profoundly changed upon mechanical injury, pathogen attack, or treatment with biotic and abiotic elicitors. Recent work has focused on the molecular genetic basis of de novo synthesis of secondary products such as phytoalexins with antibiotic properties and lignin or lignin-like compounds for cell wall reinforcement (Lamb et al., 1989; Dixon and Harrison, 1990; Walter, 1992). A het- erogenous class of defense-related proteins termed pathogenesis-related proteins that includes hydrolases and a number of functionally unidentified proteins is also under extensive study (Walter et al., 1990b; Cutt and Klessig, 1992). However, molecular alterations in primary metabo-

This work was supported by h n d s from the Commission of the European Communities (ECLAIR, AGRE 0021) awarded to M.H.W., by the Vater und Sohn Eiselen Stiftung, Ulm, Germany, and the Bundesministerium fiir Forschung und Technologie, Ger- many, awarded to D.H. J.S. acknowledges financia1 support from these organizations for predoctoral fellowships. M.H.W. was the recipient of a research position donated by the Eiselen Stiftung during part of this work.

* Corresponding author; e-mail waltermhOruhaix1 .rz.uni- hohenheim.de; fax 49-71 1-459-3751.

lism that presumably provide building blocks and energy for the biosynthesis of defense compounds have received much less attention. Fluctuations in the levels of malate and changes in activities of malate-transforming enzymes upon stress conditions of any nature suggest a role of malate metabolism in plant defense (Levitt, 1980).

Malate is known to be involved in a wide variety of primary physiological processes, including the tricarboxy- lic acid (Krebs) cycle, the glyoxylate cycle, C, and CAM photosynthesis, and control of intracellular pH (Lance and Rustin, 1984). It can be shuttled between cellular compartments and stored in plant vacuoles to a considerable extent (Gietl, 1992; Gout et al., 1993). Malate oxidation is accom- plished through various isoforms of MDHs (EC 1.1.1.37) and MEs (malate dehydrogenases [decarboxylatingl, EC 1.1.1.38-EC 1.1.1.40), which are active in different cellular compartments, including cytosol, mitochondria, and chloroplasts (Edwards and Andreo, 1992; Gietl, 1992; Ivanish- chev and Kurganov, 1992). Malate has been characterized as a storage molecule for reducing equivalents and CO,, and one or both of these are released by the action of MDHs or MEs, respectively (Lance and Rustin, 1984).

NADP-ME (EC 1.1.1.40) catalyzes the oxidative decarboxylation of L-malate to yield pyruvate, reducing equivalents (NADPH), and CO, (Wedding, 1989; Edwards and Andreo, 1992). NADP-ME acts in many different metabolic pathways in both animals and plants. In mammalian liver, it is a major NADPH-generating enzyme for lipogenesis (Bagchi et al., 1987). In plants, NADP-ME is well known for its prominent role in the specialized mode of photosynthesis in NADP-ME-type C, plants such as maize, sugar cane, and sorghum (Hatch, 1987). In these C, species an abun- dant isoform is localized in the chloroplasts of bundle sheath cells, where it releases CO, from malate to be used in carbon fixation by ribulose-1,5-bisP carboxylase. In CAM plants, a cytosolic isoform also functions as a decarboxyl- ase of malate to donate CO, for the Calvin cycle (Ting, 1985). Apart from these specialized uses, cytosolic forms of NADP-ME seem to be present in a11 plants and fulfill diverse housekeeping functions due to their universal pres- ente in many different tissues. These other, nonphotosyn-

Abbreviations: AdoMet, S-adenosylmethionine; CaMV, cauli- flower mosaic virus; MDH, malate dehydrogenase; ME, malic enzyme; 4-MU, 4-methylumbelliferone; SOD, superoxide dismutase; X-gluc, 5-bromo-4-chloro-3-indolyl 6-D-ghcuronide.

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950 Schaaf et al. Plant Physiol. Vol. 108, 1995

thetic roles are less well understood. They may be anaple- rotic, providing NADPH and pyruvate in biosynthesis, or catabolic, participating in respiration of these products (Edwards and Andreo, 1992).

The primary structure of severa1 plant NADP-MEs is now known from cDNA studies and has led to the identi- fication of a NADP-ME cDNA clone from common bean (Phaseolus vulgaris L.), which was earlier erroneously be- lieved to encode another NADP-dependent dehydrogenase, a cinnamyl-alcohol dehydrogenase (Walter et al., 1988, 1990a, 1994). The deduced amino acid sequence of this cDNA, now redesignated cPvMEl, displays 73 to 79% sequence identity without gaps to other plant NADP-MEs, clearly demonstrating its true identity as a bean ME clone (Walter et al., 1994). The PvMEl protein does not contain an apparent signal peptide for translocation into chloroplasts, which strongly suggests that it represents a cytosolic isoform of NADP-ME. This is in accordance with its origin from a C, species, which is considered to contain genes for cytosolic forms but not for chloroplastic NADP-MEs. More- over, the N terminus contains none of the features described for mitochondrial targeting peptides (Von Heijne et al., 1989; Walter et al., 1994).

We have recently isolated the gene corresponding to the cPvMEl clone and have determined its exon/intron structure as well as approximately 2.7 kb of 5' flanking promoter sequences (Walter et al., 1994). As shown earlier, transcription of the PvMEl gene is rapidly activated by funga1 elicitor treatment in bean cell-suspension cultures (Walter et al., 1988). To further analyze defense-related expression patterns of this gene we have generated transgenic tobacco plants expressing the GUS reporter gene under the control of the PvMEl gene promoter. Here we report that the PvMEl promoter directs both developmentally regulated, tissue-specific expression of an unexpected nature and inducible gene activity stimulated by environmental factors known to up-regulate other plant defense genes. Studies of a CaMV 35s promoter-driven GUS gene in tobacco and RNA blot analysis of bean plants indicate that these promotor features do not result from artifacts introduced by the expression of the gene fusion in the heterologous host plant but reflect the situation in the promoter donor plant.

MATERIALS A N D METHODS

Gene Fusion Constructions

The PvMEl promoter fragment was excised from a 4.1-kb HindIII genomic DNA fragment (Walter et al., 1994) by HindIII and BcZI digestion. Because BcZI cleaves immediately 5' to the ATG translation start codon, the resultant fragment of 2826 bp contains, in addition to promoter regions, the entire leader sequence, including a small intron. A transcriptional fusion of this fragment (-2664 PvMEl promoter fragment) to the GUS gene was generated by ligating it into compatible ends of a HindIII/BamHI-cut binary vector, pBI 101.4 (a kind gift of M. Bevan). pBI 101.4 (Bevan et al., 1989) contains a modified ATG translation start site vicinity for the GUS gene optimized according to

Kozak's rule and an nptII gene for kanamycin selection of transformants. To produce a comparable GUS fusion with the CaMV 355 promoter, an 800-bp HindIII/BamHI fragment excised from pBI 121 (Jefferson, 1987) was inserted into the appropriately cut vector pBI 101.4. The fusions were verified by restriction analysis and by DNA sequenc- ing of fusion borders using a GUS-gene-specific primer (Clontech, Palo Alto, CA).

Plant Transformation and Growth

The plasmids containing the chimeric PvME1-GUS genes were transferred from Escherichia coli HBlOl to Agrobacte- rium tumefaciens LBA4404 by the direct transformation method. Transformed agrobacteria were analyzed for the presence of intact, unrearranged T-DNA by DNA gel blot analysis. Leaf disc transformation of Nicotiana tabacum cv Petit Havana SR1 and regeneration of transgenic plants were performed according to standard procedures (Horsch et al., 1985). Primary transformants were maintained axenically at 20°C under a 16-h light (7000 lux)/Bh dark cycle on half-strength Murashige and Skoog medium (pH 5.8) containing 100 pg mL-' kanamycin. The integrity and number of T-DNA copies inserted into the tobacco genome were estimated by DNA gel blot analysis. Tobacco genomic DNA samples were cut with HindIII + EcoRI or HindIII. The blots were hybridized with a ,'P-labeled GUS-specific probe (SmaI-SstI fragment from pBI 101.4). Primary transformants were also grown to maturity in soil in a greenhouse to produce seed. Seeds were germinated on the same medium as was used for the primary transformants after surface sterilization by a 7% (w/v) sodium hypochlorite solution. Developmental patterns of promoter activity were analyzed in To and TI plants and have now been corroborated in T, plants (data not shown).

Stress Treatments

Tissues of axenically grown primary transformants (To) 3 to 4 weeks after subculture were used. Intact tissue was immediately snap frozen in liquid nitrogen upon harvest and kept as untreated controls (basal GUS activity). For wounding conditions, detached leaves, stems, and roots were cut into 1- to 2-mm slices with a sterile razor blade and aged for 40 h at 25°C on filter paper moistened with 20 mM sodium phosphate buffer (pH 5.8). The material was then snap frozen and kept at -7O"C, like the control tissue. For the application of potential inducers, the respective compounds were added to the phosphate buffer and the wounded tissue was moistened with the resultant solution under the conditions described above. The compounds used were purchased in pure or ultra-pure quality from Serva (Paramus, NJ) (GSH, GSSG, and mercuric chloride), Sigma (NADPH [sodium salt]; ascorbic acid [sodium salt]; cellulase [Trichoderma viridel; catalase [bovine liverl; and phytohormones), or Aldrich (H'O,). The elicitor from Col- Zetotrichum lindemuthianum (a kind gift of C.J. Lamb and co-workers) was the high molecular weight fraction heat released from isolated mycelial walls (Anderson-Prouty and Albersheim, 1975). For histochemical analysis, leaf

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Malic Enzyme

discs (8 mm diameter) were cut with a cork borer from fully expanded leaves and exposed to treatment solutions. Wounding of intact detached leaves was done by pricking with an Eppendorf tip. GSH application was achieved by covering the wound site with a filter paper soaked with 10 mM GSH in phosphate buffer. Discs and intact leaves were incubated in the dark for 30 h and rinsed with 50 mM sodium phosphate buffer (pH 7.0) prior to histochemical staining for GUS activity. GSH treatment of seedlings was achieved by staining in a X-gluc staining solution supplemented with GSH in a final concentration of 10 mM.

GUS Assays

GUS activity in crude protein extracts was determined fluorometrically as described (Jefferson, 1987) except that the extraction buffer contained methanol in a final concentration of 20% (v/v) to reduce background activity that might be caused by endogenous GUS activity of plant material (Kosugi et al., 1990). Fluorescence of 4-MU produced from the corresponding glucuronide through GUS activity was measured on a Hoefer (San Francisco, CA) DNA fluorometer TKO 100. Data are presented as the mean ? SD of GUS activities from three independent experiments. Protein content was determined by the Bio-Rad Protein Assay Dye Reagent and BSA as a standard.

Histochemical staining for GUS activity was done essen- tially as described (Jefferson, 1987) using X-gluc (Biosynth AG, Staad, Switzerland, or Clontech) as a substrate. Hand sections and intact organs were vacuum infiltrated for a few minutes with 1 mM X-gluc in 50 mM sodium phosphate (pH 7.0) and 0.1 to 0.5 mM each of K,[Fe(CN),] and K,[Fe(CN),] and incubated at 37°C for 6 to 18 h. For staining floral organs, 0.2% (w/v) PVP and 10 mM DTT were included in the substrate solution to reduce tissue brown- ing. Leaf discs, whole leaves, and seedlings were stained in the presence of 0.01 to 0.02% Triton X-100. Stained tissue was cleared of Chl by washes in a series of aqueous ethanol (35, 50, 70, and 80% [v/vl) prior to visual analysis in a microscope (stereo microscope IV B or photomicroscope 11, Zeiss). Thicker stem sections (2-3 mm) used for microtome sectioning were pretreated as described by Hauffe et al. (1991).

Embedding was performed as follows. The stained and dehydrated tissue was infiltrated twice by equal parts of methacrylate and 2-hydroxyethyl-methacrylate for 6 h and overnight, respectively. Subsequently, the same procedure was done for infiltration of solution A (60 parts 2-hydroxyethyl-methacrylate, 20 parts methyl methacrylate, 16 parts ethylene glycol monobutyl ether, and 2 parts PEG 400 supplemented by 270 mg/100 mL benzoyl peroxide and 1 part N,N-dimethylaniline prior to usage). The material was polymerized at 4°C for 2 to 3 d and 10- to 20-pm sections were cut on a rotary microtome (model No. 2040, Jung, Heidelberg, Germany). The sections were incubated with water on cleaned slides and dried at 40°C. The sections were embedded in immersion oil, and photographs were taken by dark-field microscopy on Kodak EPY 64T or Ek- tachrome 160T slide films.

Gene Regulation 951

RNA Gel Blot Analysis

Total RNA was prepared from leaves of 3- to 4-week-old P. vdgaris L. cv Processor plants grown in the greenhouse. Treatments were performed by immersing the leaf stalks connected to intact young, fully expanded leaves into 10 mM GSH or 10 mM salicylic acid solutions in 20 mM sodium phosphate buffer (pH 5.8). RNA isolated from leaf blades was fractionated by electrophoresis in a formaldehyde- denaturing gel and blots were hybridized with the labeled cPvMEla cDNA probe (Walter et al., 1994) in 1 M

NaCI, 1% (w/v) SDS, 10% (w/v) dextran sulfate, and 100 pg mL-' salmon sperm carrier DNA at 65°C overnight. Final washes were in 0.1 X SSC, 0.5% (w/v) SDS, at 65°C for 30 min.

RESULTS

Construction of a PvMEl Promoter-GUS Fusion and Tobacco Transformation

A transcriptional fusion of the available PvMEl promoter was created by ligating a 2.8-kb HindIII-BclI fragment containing 2.6 kb of promoter sequences and the entire untranslated leader, including a small leader intron (Walter al., 1994) into the promoterless GUS vector pBI101.4. This construct (-2664PvME1-GUS) was introduced into tobacco, and plantlets were regenerated under kanamycin selection. Likewise, control plants transformed with promoterless pBI 101.4 or with pBI 101.4, in which the GUS gene is driven by the CaMV 35s promoter, were generated. Severa1 -2664PvMel-GUS transformants that were analyzed for organ-specific expression in preliminary experiments gave appreciable GUS activity only in stem extracts. Twenty regenerated kanamycin-resistant -2664PvME1-GUS transformants were then screened for GUS activity in stem extracts, and 19 exhibited appreciable activity. Nine plants with moderate to high activity were chosen for further analysis. Southern blot analysis revealed the presence of three to five unrearranged T-DNA copies in the genome of these transformants (data not shown).

Organ-Specific Expression of the -2664PvME1 -GUS Fusion

The expression of the -2664PvME1-GUS fusion in stems, roots, and leaves of nine independent transformants was analyzed by fluorometric assays of extractable GUS activities. As shown in Figure 1, developmental expression was highest in stems. Moderate to low levels of GUS activity (2- to 27-fold lower than in stems; 13-fold on average) were found in roots. In leaves, GUS expression was barely detectable except for in the high-leve1 expressor plant MEl- GUS16. Although the absolute levels of expression varied, as expected, between independent transformants, the same overall pattern was seen in a11 of them: stems > roots > leaves. In contrast, six plants harboring the CaMV 35s-GUS fusion displayed similar levels of GUS activity in each of the vegetative organs (Fig. 1). GUS activity in control plants containing the promoterless GUS gene was at the detection limit (0-2 pmol 4-MU min-' mg-' protein).




h

ME ME ME ME ME ME ME ME ME CaMV CaMV CaMV CaMV CaMV 4 1 12 23 10 19 14 20 16 14 1 23 11 9

Figure 1. Developmental expression of the -2664PvME1 -GUS gene fusion in vegetative organs of transgenic tobacco plants. GUS activity was determined by the fluorometric assay from plantlets harboring the -2664PvME1 promoter-GUS or the CaMV 35s promoter-GUS (control) constructs 3 to 4 weeks after rerooting. Individual transformants are identified by numbers. Fully developed young leaves, the upper half of the stem, and all of the root material were analyzed. Note different scales for data presentation.

Spatial Patterns of PvMEl Promoter Activity

The developmentally regulated, tissue-specific pattern of PvME1-GUS expression in vegetative and reproductive organs was investigated by histochemical staining. The analysis was performed on several transformants of the -2664PvME1 promoter-GUS fusion with high expression levels, and the results obtained from transformants MEl- GUS16 and ME1-GUS20 are shown in Figure 2. Staining of transverse and longitudinal hand-cut sections revealed that the strong GUS activity in stems was confined to cells of the vascular cylinder and the pith parenchyma. Even after prolonged incubation, no staining was detectable in other stem tissues such as the cortex or the epidermis (Fig. 2, D and E). Sections of node regions also exhibited GUS activity at the leaf trace and at the base of axillary buds (not shown).

To identify the specific cells expressing the PvME1-GUS construct in vascular tissue, microtome sections of histo- chemically stained and plastic-embedded stems were prepared and analyzed in dark-field microscopy. On these dark-field micrographs, the indigo product of GUS enzyme activity appears as purple-red staining. The microtome sections revealed the presence of GUS activity in cells of the developing xylem and the interna1 phloem, whereas no staining was seen in the external phloem (Fig. 2F). In addition, these sections confirmed the expression of GUS activity in the pith parenchyma and its absence in the cortex. The staining shown in Figure 2, A to C, demon-

strates that plants expressing the reporter gene under the control of the CaMV 35s promoter did not show a similarly confined GUS staining pattern. We have thus shown that the absence of staining in particular tissues of PvME1-GUS transformants was not due to artifacts caused by the lack of substrate accessibility or by differences in metabolic activity of these cells but rather reflects cell-specific activity of the PvMEl promoter.

Whereas the fI uorometrically determined PvMEl-GUS expression in roots and leaves turned out to be too weak for histochemical localization, strong GUS staining could be observed in several tissues of floral organs and reproductive structures. The results are presented in Figure 2, G through J. In flower buds about 5 mm in length that were hand sectioned longitudinally through the ovary, GUS activity was present throughout parts of the ovary, the pistil, the stigma, and the anthers (Fig. 2G). Highest GUS expression was detected in the receptacle. Sepals and petals did not show GUS activity except for vascular bundles. In transverse sections through the anthers of 5-mm buds, intense blue staining was observed in the vascular cylinder, the stomium, and the tapetal cells (Fig. 2H). Cross-sections of immature seed pods exhibited GUS activity in the ovary wall, the immature seeds, and the vascular bundles of the placenta (Fig. 21). Embryos of mature seeds also showed strong expression of GUS (Fig. 2J). Notably in embryos, GUS activity appeared to be absent from the dista1 ends of the root axis and of the cotyledons, whereas cells of the provascular cylinder were stained more intensely.

Figure 2. (On facing page.) Histochemical localization of GUS activity in vegetative and reproductive tissues of transgenic tobacco plants. GUS activity was detected by staining of ME1-GUS16 (G, H, I, K, N, O), MEl-GUS2O (D, E, F, L, M), and CaMV-GUS23 (A, B, C) transformants with the chromogenic substrate X-gluc. A to C, CaMV-GUS expression in young stems in hand-cut transverse (A) and longitudinal sections (B) (magnifications 20X) and in a microtome transverse section (magnification 150X) viewed in dark field (C). D to F, PvMEl -GUS expression in young stems in sections corresponding to the CaMV-GUS analysis (A-C). G to j, PvME1 -GUS expression in reproductive tissue. G, Young flower (5." bud) split longitudinally ( 7 ~ ) . H , Dark-field view of a transverse section of an anther (5-mm bud) with tapetal cells staincd (35X). I, Transverse section of a seed pod ( 7 ~ ) . J, lntact embryo (1 lOX). K to O, lnduction of PvME1-GUS expression by external stimuli. K, Whole leaf wounded (W) or wounded and treated with 10 mM GSH (WG). L and M, Young seedlings stained for GUS activity (L) or treated with 10 mM GSH in the staining solution (M). N and O, Leaf discs incubated for 30 h on filter paper moistened with phosphate buffer without (N) or with the addition of 60 wg/mL funga1 elicitor (O). www.plantphysiol.orgon August 14, 2020 - Published by Downloaded from

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Malic Enzyme Gene Regulation 953

M OFigure 2. (Legend appears on facing page.)



954 Schaaf et al.

Stress Activation by Wounding and Clutathione in- Different Organs

The vegetative organs of transgenic plants harboring the -2664PvME1 promoter construct were subjected to stress treatments known to induce the expression of other bean defense genes (Wingate et al., 1988; Liang et al., 1989a). To provoke a wounding response, detached leaves, roots, and stems of six individual transformants were cut into 1- to 2-mm slices and incubated on filter paper moistened with a phosphate buffer solution (pH 5.8) for 40 h. The pH conditions correspond to those maintained in the nutrient agar of sample plants. GUS activity was determined by the fluorometric assay. Relative to intact tissue, wounding caused a marked increase in GUS activity (Table I). De- pending on the individual transformant and the organ analyzed, a 2- to 8-fold induction was observed. Leaf tissue with very low basal levels of GUS activity exhibited on average a 4.2-fold increase upon wounding. In root and stem tissue with higher levels of background developmental expression, the wound response involved a 3.6- and 1.8-fold increase, respectively.

The effect of GSH on wounded tissues was examined by adding GSH in a final concentration of 10 mM to the buffer solution during the 40-h incubation period. Compared to GUS activity after wounding alone, the GSH treatment of leaves resulted in a further average increase of GUS expression by a factor of 6.8 and in an 8- to 70-fold (average 31-fold) induction relative to untreated tissue. Application of GSH to sliced roots and stems led to only 2- and 1.5-fold increases in extractable GUS activity, respectively.

To rule out the possibility that a drop in the pH of the buffer solution brought about by the addition of GSH was responsible for the observed effects, we assayed in leaf tissue a buffer solution adjusted to the pH value of the GSH/buffer solution (pH 3.6). We also adjusted a GSH/ buffer solution to the pH of the original buffer without GSH (pH 5.8). The strong acidic conditions without GSH caused only a slight (1.8-fold) increase in GUS activity relative to wounded tissue, compared to 6.5-fold induction by GSH under the same pH conditions. However, adjust- ment of the GSH solution to pH 5.8 abolished most of the

Plant Physiol. Vol. 108, 1995

activating potential of GSH (2.3-fold induction), indicating that GSH can exert its inducing effect only under appro- priate pH conditions (data not shown).

To further prove that the increase in extractable GUS activity observed in the experiments described above was brought about by features of the PvMEl promoter and not by alterations in GUS enzyme stability and/or activity by the inducing treatment, controls were performed with CaMV 35s-GUS transformants. As shown in Table I1 for four representative plants transgenic in the CaMV 35s-GUS construct, the treatments did not affect the level of GUS activity to a considerable extent. A slightly elevated level of extractable GUS activity in GSH-treated controls may have been caused by the antioxidant properties of GSH mediat- ing a stabilization of enzyme activity, but it does not chal- lenge the GSH-inducible features of the PvMEl promoter in tissues with low developmental background expression levels, such as leaves. Taking possible effects of stimuli on the GUS enzyme into account, we have also included controls of CaMV 35s-GUS transformants in the subsequent screening experiments for nove1 inducing agents (see below).

Screening for lnducers in Leaf Tissue

Because of the very low basal-leve1 activity of leaf material, this tissue appeared best suited for additional induction experiments. Three representative PvMEl-GUS transformants (ME1-GUS19, ME1-GUS23, ME1-GUS20) and two CaMV 35s-GUS control plants (CaMV-GUSI 1, CaMV- GUS23) were selected. The stimuli were applied in the phosphate buffer solution to sliced leaf tissue. The absolute figures of GUS activity after wounding and the various simultaneously acting stimuli are summarized in Table 111. GSH turned out to be the strongest inducer, giving a 7.5- or 4.9-fold increase on average over wounding alone for 10 mM or 5 mM applied, respectively. We then demonstrated that GSSG is also active in eliciting PvMEl promoter activity, albeit with reduced strength, causing 5.4- or 3.4-fold increases using concentrations of 10 mM or 5 mM, respectively. Cellulase Onozuka R10 (0.5 mg/mL), a commercial crude enzyme preparation from T. viride, was another

Table 1. Stress activation of PvME1-GUS expression by wounding and GSH in different organs Data are presented as the mean 2 SD of CUS activities from three independent experiments.

GUS Activity in PvME1-GUS Transformant

4 19 23 14 20 16 Treatment

Leaf Untreated 3 t l Wounded 9 2 1 Wounded + GSH 74 2 2

Untreated 31 2 1 0 Wounded 114 t- 49 Wounded + GSH 188 ? 38

U ntreated 294 2 106 Wounded 734 2 170 Wounded + CSH 1,291 2 457

Root

Stem

6 2 1 1 8 ? 4

1 1 2 2 16

73 t 14 266 t 90 513 IT 156

1,952 t 661 3,914 IT 280 5,172 -C 570

pmol4-MU

6 2 2 5 0 2 16

423 2 64

113 2 38 3 3 4 t 113 667 2 130

1,267 t 408 2,523 2 766 3,275 ? 123

min- ’ mg- ’ protein

9 2 1 41 2 8

226 2 14

1 5 6 2 41 460 2 129 899 2 184

3,211 2 464 4,704 2 966 6,398 t 1,006

18 t 8 87 ? 21

671 t 95

266 t 26 1,281 2 303 2,568 ? 261

5,500 2 661 8,266 2 513

12,141 t 917

347 t 123 601 t 106

2,789 t 876

679 2 99 2,442 t 539 3,880 t 11 1

14,276 t 1,161 20,831 t 3,348 28,512 t 1,938




Table II. Effect o f inducina treatments on CaMV 35s-GUS exmession in different organs

C U S Activity in CaMV 35s-CUS Transformant

14 1 31 11 Treatment

Leaf U ntreated Wounded Wounded + GSH

U ntreated Wounded Wounded + GSH

Untreated Wounded Wounded + CSH

Root

Stem

7,840 t 1,890 10,710 t 2,110 19,730 2 3,190

16,550 t 2,280 22,070 2 4,320 25,010 t 3,220

10,200 ? 2,520

14,360 t 2,000 I 2,080 2 2,180

pmol 4-MU min-’ mg-’ protein

32,180 t 5,560 29,980 t 4,7i o

49,510 rt 8,240 62,570 t 13,260

42,442 t 5,180 i 05,4i o t 4,480

27,590 t 6,740 43,700 ? 5,340 60,390 t 14,430

85,910 t 7,440 80,050 t 12,520

1 1 0,620 t 24,710

45,300 ? 6,240 45,400 2 9,580 49,030 t 8,330

61,450 t 2,890 83,l O0 t 21,660

102,134 t 9,280

55,320 ? 9,600 51,970 ? 11,370 99,240 2 10,310

85,960 t 5,790 92,700 t 23,280 80,930 t 15,290

77,390 ? 16,010 87,360 2 18,940

i i 6,300 2 8,890

strong inducer, virtually as potent as GSH (7.1-fold, Table 111). Boiling for 20 min almost completely abolished the elicitor potential of the cellulase. Ethylene, administered as the ethylene-releasing compound ethephon, can also be listed among the strong inducers of PvMEl promoter activity (6.1-fold). Furthermore, elicitor from the bean funga1 pathogen C. lindemuthianum, which had earlier been shown to cause a transient accumulation of PvMEl mRNA in bean cell cultures (Walter et al., 1988), was included in the induction assays. Here we have applied three different concentrations, a11 of which resulted in significantly elevated extractable GUS activity (2.8- to 4.6-fold). A some- what weaker inducing effect (2.4-fold) was exerted by mercuric chloride.

To get clues on a possible regulation of the PvMEl promoter by redox perturbations, we applied ascorbic acid (sodium salt) and NADPH (reduced form) in the concentrations given in Table 111. NADPH gave a 3.5-fold induc-

tion and ascorbic acid caused a 1.9-fold increase in GUS activity. Addition of catalase, an H,O,-destroying compound, resulted in a considerable (3.7-fold) induction over the wounding level. On the contrary, H,O, itself (0.1 and 1% [v/vl) did not have an up-regulating effect on GUS expression (data not shown).

Additional compounds found to be ineffective in inducing promoter activity under the conditions used are severa1 phytohormones (naphthalene acetic acid [ l O O WM], 2,4-dichlor- phenoxy acetic acid [10 WM, 100 pM], ABA [I0 WM, 100 pM])

and salicylic acid (2 mM, 5 mM, 10 mM). Salicylic acid appeared to have damaging effects on the integrity of the plant material, at least in higher concentrations (data not shown). Except for a minor effect of GSH, the stimuli tested did not cause induction in the CaMV 35s-GUS controls (Table 111).

A possible inducibility by light was analyzed in intact seedlings, which were dark adapted for 72 h and then exposed to white light for 24 h or further kept in the dark

Table 111. Screening for inducers in leaves expressing PvMEI-GUS

C U S Activity

PvMEl -CUS CaMV 35s-GUS (X10’) Treatment

19 23 20 11 23

U ntreated Wounded Wounded + treatment

Ascorbic acid (1 O mM)

Mercuric chloride (100 p ~ ) Catalase (1 ?‘O [w/v]) NADPH (5 mM) Fungal elicitor (60 pghnL) Fungal elicitor (600 pg/mL) Fungal elicitor (1200 pg/mL) Ethephon (1 mg/mL) Cellulase, active (0,5 m g h L ) Cellulase, inactive (0,5 mg/mL) GSH (5 mM) GSH (10 mM)

GSSG (5 mM)

GSSG (10 mM)

a n.d., Not determined.

pmol4-MU min- ’ mg- protein 6 t l 6 2 2 18 t 8 553 2 97 495 2 a2

1 8 t 4 5 0 ? 16 87 rt 21 540 2 67 576 t 113

32 2 5 39 t 15 68 2 11 33 t 5 56 t 11 63 t 15 79 ? 2 72 t 17

115 2 31 25 2 5 70 t 11

112 t 16 58 t 20 93 ? 13

102 t 22 1 4 0 2 7 226 2 32 269 t 30 114 2 26 186 t 17 254 t 88 388 t 52 379 2 120 81 2 1 3

194 t 29 423 ? 64 175 ? 55 291 t 37

171 t 6 0 202 2 9 246 t 68 297 -+ 21 272 t 87

378 t 129 571 rt 87

121 2 14 594 2 16 671 ? 95

464 2 117

383 rt 58

629 rt 185

288 2 92

530 ? 49 625 ? 34 534 ? 45 572 t 24 392 2 31 322 2 29 597 t 48 500 t 63

n.d.” n.d. 581 t 48 573 2 73

n.d. n.d. 525 2 38 473 2 23 540 ? 120 658 ? 51 487 2 125 621 ? 65

992 2 103 1054 2 45

689 t 93 790 t 75

n.d. n.d.

n.d. n.d.




GSH SA

024 8 12 24 12 0 2 4 8 12 2412

kbh2.2

Figure 3. Accumulation of PvMEl mRNA in bean plants duringtreatment with GSH or salicylic acid. An RNA gel blot of bean totalRNA probed with the cPvMEla cDNA fragment is shown. RNA wasobtained from bean leaves, the leaf stalks of which were immersed in10 rriM GSH or 10 ITIM salicylic acid (SA) dissolved in phosphatebuffer (pH 5.8) for the times indicated (0-24 h). Controls incubatedin buffer only for 12 h (C 12) were included. The size of the expectedPvMEl transcript is given in kb.

for the same period. No effect of the light treatment wasseen (data not shown).

The inducing effects of GSH and the Colletotrichum elic-itor were further investigated by histochemical staining.Whereas GUS-positive staining was hardly detected in de-tached whole leaves after wounding alone, the applicationof 10 mM GSH to a wound site caused a strong induction ofGUS activity that was notably restricted to the site of GSHtreatment and the adjacent vascular tissue (Fig. 2K). Wealso demonstrated that expression of the PvMEl-GUS fu-sion was inducible in cotyledons of 8-d-old seedlings. Inuntreated seedlings, GUS staining was detected only in themain vein of the cotyledons (Fig. 2L), whereas in GSH-treated seedlings, the primary leaves yielded strong GUS-positive staining, which, however, excluded the radicle(Fig. 2M). Finally, discs were cut from intact leaves andincubated on filter paper moistened with phosphate buffer.GUS activity was preferentially expressed in leaf veins, anda faint staining was spread irregularly over the whole leafdisc (Fig. 2N). A staining pattern that was basically thesame but more intense appeared if the solution was sup-plemented with the Colletotrichum elicitor (Fig. 2O).

PvMEl mRNA Accumulation in Bean Plants

Promoter-GUS fusions in transgenic plants are not suitedfor time-course studies on gene activation by external stim-uli, since stability of GUS mRNA and GUS protein areindependent of the respective characteristics of the productof the original coding sequence driven by the promoter.Moreover, basic characterization of expression patterns byhybridization analysis can avoid misinterpretations causedby occasionally observed GUS artifacts (Uknes et al., 1993).For these two reasons, a time-course study on the accumu-lation of PvMEl mRNA in bean plants was initiated. Ap-plication of 10 mM GSH and 10 mM salicylic acid to slicedbean leaves under buffer conditions used in tobacco led toobvious tissue damage and poor quality of RNA prepara-

tions. Therefore, a different method of stimulus applicationwas performed for the bean leaves by immersing the leafstalks of intact leaves into the inducing solutions. Feeding10 mM GSH in such a way resulted in strong induction ofPvMEl mRNA in the leaf (Fig. 3). Four hours after onset ofGSH feeding, PvMEl mRNA started to accumulate andreached high levels after 12 to 24 h. Feeding of salicylic acid(10 mM) caused a delayed and less-pronounced inductionrelative to GSH treatment, but inducing effects of salicylicacid could be recognized, in contrast to the results intobacco, under different conditions of stimulus application.The evidence for the strong inducing effects of GSH onPvMEl mRNA accumulation in bean is in good agreementwith the results from the PvMEl promoter characterizationin transgenic tobacco.

DISCUSSION

We have investigated in transgenic tobacco the regula-tory characteristics of a bean promoter that in its nativemetabolic environment drives the expression of cytosolicME-coding sequences. Due to the lack of a routine proce-dure for bean transformation, we have performed the anal-ysis of promoter-GUS fusions in the heterologous hosttobacco. Several studies of bean promoter-GUS fusions intransgenic tobacco indicate that expression of the reportergene in this host is regulated in a manner similar to that inthe promoter donor plant (Liang et al., 1989a, 1989b;Schmid et al., 1990). Nevertheless, we have addressed theproblem of possible GUS artifacts in two ways. First, wehave shown that the responsiveness of the promoter tofungal elicitor and glutathione in tobacco is paralleled bytranscriptional activation of the PvMEl gene and/or accu-mulation of PvMEl mRNA in bean cell cultures and plantsby the same stimuli (Walter et al., 1988) (Fig. 4). Further-more, RNA preparations from different tissues of etiolatedbean seedlings gave a hybridization signal for PvMElmRNA only with epicotyl tissue comparable to the strongPvMEl promoter activity in tobacco stems (M.H. Walter,unpublished results). Second, we have included controlplants in our analyses that express the GUS gene under thecontrol of the strong constitutive CaMV 35S promoter.These control plants displayed promoter features very dif-ferent from the PvMEl promoter, indicating that the de-scribed developmental and defense-related expression pat-terns are established by specific properties of the PvMElpromoter.

Ascorbate

\Glutathione(oxid.) GSSG

NADPHNADP"

Ascorbate- Dehydroascorbate-Peroxidase Reductase

\(Glutathione-Reductase

\ \Dehydroascorbate Glutathione NADP

Figure 4. The Halliwell-Asada pathway (adapted from Bowler et al.,1992). www.plantphysiol.orgon August 14, 2020 - Published by Downloaded from

Copyright © 1995 American Society of Plant Biologists. All rights reserved.



It is possible that the 5’ untranslated leader region of the PvMEl gene included in our construct contributes to the observed promoter activity patterns by conferring features of message stability or translatability to the GUS gene. If operative, this would give an improved representation of the regulatory situation in bean compared to the fusion of the promoter alone. In addition, we have taken into account possible effects of externa1 stimuli on GUS enzyme activity and stability. Therefore, we have as extensively as possible ruled out GUS artifacts observed occasionally at the levels of GUS gene expression (Uknes et al., 1993) or the GUS histochemical staining assay (De Block and Debrou- wer, 1992) by these control experiments.

Tissue-Specific Promoter Activity and lmplications for Nove1 Developmental Roles of Cytosolic ME

NADP-ME has clearly defined roles as an enzyme releasing CO, from malate for refixation by Rubisco in certain C, and CAM plants. The genes encoding C, pathway enzymes have undergone evolutionary modification and/or dupli- cation for their use in the specialized modes of photosynthesis. Ancestor genes for cytosolic isoforms of NADP-ME are still present and functional in these plants as well as in other plants, other eukaryotes, and prokaryotes. The housekeeping functions of cytosolic NADP-ME in plants are only poorly defined. Suggested roles include a participation in fruit ripening by promoting deacidification and gluconeogenesis, a contribution to the maintenance of intracellular pH, general anabolic functions by providing NADPH and pyruvate for biosynthetic reactions, and catabolic functions by channeling NADPH and pyruvate into respiratory pathways for energy production (Edwards and Andreo, 1992).

In our analysis of the PvMEl gene promoter, we have demonstrated very low constitutive expression and lack of light inducibility in leaves, consistent with its origin from the C, plant P. vulgaris (Fathi and Schnarrenberger, 1990). We have observed moderate to high activities in roots and in reproductive structures, in particular in fruit and seed. A striking nove1 feature of developmentally regulated NADP-ME gene activity was the strong expression in stems, which was confined to the vascular tissue and the pith parenchyma. A similar and likewise unexpected cellular preference of a housekeeping plant gene for the vas- culature has been observed for the AdoMet synthetase (Peleman et al., 1989). AdoMet synthetase acts as a methyl group donor in a11 kinds of organisms (Peleman et al., 19891, and NADP-ME acts to donate NADPH in nonpho- tosynthetic tissues of plants and in nonplant species (Fab- regat et al., 1987; Edwards and Andreo, 1992). On the basis of its vascular expression, a role for AdoMet synthethase in providing methyl groups for lignification has been pro- posed (Peleman et al., 1989). It is conceivable that NADP-ME expression in xylem is also associated with lignin biosynthesis by providing NADPH for the two NADPH-dependent reductive steps in monolignol biosynthesis (Walter, 1992). This hypothesis is consistent with preliminary results from a down-regulation study by NADP-ME antisense RNA expression in transgenic tobacco

plants showing reduced deposition of lignin (Schuch et al., 1990) and with the induction of NADP-ME mRNA in dif- ferentiating bean calli undergoing lignification (Grima-Pet- tenati et al., 1989). The provision of NADPH for lignin biosynthesis has been attributed to the oxidative pentose phosphate cycle (Pryke and ap Rees, 1977). However, in rat liver undergoing lipogenesis, activation of the pentose phosphate cycle is supplemented by recruitment of ME to meet the large need for NADPH in these particular developmental circumstances (Fabregat et al., 1987). A similar situation might exist in lignin-synthesizing plants.

Our hypothesis does not explain the considerable promoter activity of the PvMEl gene in the pith tissue of stems or in reproductive tissues. Since NADP-ME is likely also to serve purposes other than lignin biosynthesis, additional, as yet undefined metabolic situations requiring NADPH may be involved in NADP-ME gene activation. Fruit- and embryo-specific expression might well be correlated with catabolic functions and energy metabolism. To determine exactly the processes in which ME gene expression is involved will undoubtedly require additional experimental approaches.

Participation of ME in Plant Defense Responses

Malate concentrations in plant cells are subject to considerable changes under a variety of physical (cold, heat, drought), chemical (herbicides, pesticides, pollutants), and pathogen stresses (Levitt, 1980; Lance and Rustin, 1984). With respect to ME, the first observation of a wounding- related decarboxylating activity for exogenously supplied malate was reported for apples sliced into discs (Rhodes et al., 1968). The development of this activity during wounding was dependent on RNA and protein synthesis. In rubber trees (Hevea brasiliensis) wounded for latex production, NADP-ME mRNA accumulated concomitantly with other defense gene transcripts (Kush et al., 1990).

We have shown here at the leve1 of promoter activity that the PvMEl gene is responsive to wounding in transgenic tobacco. Leaf tissue, which exhibited only very low basal levels of GUS activity, did respond in a more pronounced fashion than other tissues. Of the defense-related stimuli applied to wounded tobacco leaves, GSH gave the strongest activity-promoting effect on the PvMEl promoter. It also caused a massive accumulation of PvMEl mRNA if fed to bean leaves. GSH has been shown to elicit a multi- component defense response similar to the reaction after funga1 elicitor treatment (Wingate et al., 1988). A possible mechanism of its action will be discussed below.

In accordance with earlier results from bean cell cultures (Walter et al., 1988), the elicitor preparation from C. lindemuthianum showed considerable activation potential in the promoter assay system. Cellulase and ethylene released from ethephon were characterized as additional strong inducers. Cellulase might act through cell-wall damage and the elicitation of subsequent repair mechanisms, including local lignification. Cellulase treatment of wounded wheat leaves caused induction of lignification (Barber and Ride, 1988). Funga1 elicitor applied to parsley cell cultures and plants caused an accumulation of mRNA for the




methyl group donor “housekeeping“ enzyme AdoMet synthetase implicated in developmental lignification (Kawal- leck et al., 1992), suggesting roles in defense-related deposition of lignin or lignin-like compounds as well.

Ethylene production accompanies many plant defense and senescence reactions. The rise in endogenous ethylene in tomato fruit at a specific stage of ripening coincides with an increase in enzyme activity of a cytosolic NADP-ME, and exogenous ethylene further enhances this activity (Goodenough et al., 1985). It has been suggested by these authors that the peak in cytosolic NADP-ME activity ac- counts at least in part for the climacteric burst in respiratory CO, release at this stage at the expense of Krebs cycle activity in the mitochondria. Such a crossover in malate utilization, giving more rapid access to NADPH for biosynthetic reactions and/or energy via an NADPH dehydrogenase at the exterior of the mitochondria, might occur in defense situations as well. Apart from the energy aspect, a considerable number of NADPH-consuming steps in plant defense reactions can easily be listed from the literature, e.g. in phytoalexin biosynthesis (Welle and Grise- bach, 1989; Fischer et al., 1990; Tiemann et al., 1991; Clem- ens et al., 1993) and in the reductase activity of a disease resistance gene (Johal and Briggs, 1992). Preliminary results from elicitor-treated alfalfa cell cultures producing isoflavonoid phytoalexins indicate that NADPH levels de- cline below 50% of controls during the first 6 h after elicitation but return to original levels after 7 to 8 h (T. Fahr- endorf and R.A. Dixon, personal communication). Such a decrease in NADPH might be the trigger for activation of NADPH-replenishing pathways including ME in plants as in animal cells (Fabregat et al., 1987).

Regulation by Redox Perturbations and Oxidative Stress

In the light of growing interest in the role of active oxygen species and oxidative stress in the regulation of plant defense reactions, we want to discuss in this context possible roles of the NADPH-supplying ME. Active oxygen species are toxic to plant cells, and protective antioxidant mechanisms to prevent cellular damage can be activated (Bowler et al., 1992). However, during pathogen attack activated oxygen species are produced rather than de- stroyed in order to kill or damage invading microbes in plants (Sutherland, 1991) and animals (Baggiolini and Wyman, 1990). Activation of oxygen involves electron transfer from a donor to oxygen as acceptor, leading to reactive oxyradical species such as superoxide and hy- droxyl radicals. At least in animals, the electron donor is cytosolic NADPH, and activated oxygen species are thought to be generated by NADPH oxidases located in the plasma membrane (Baggiolini and Wyman, 1990). Super- oxide radicals are converted into H,O, by ubiquitously distributed SODs (Bowler et al., 1992)

Oxidative stress is thought to alter the cellular redox toward a more oxidized status. Surprisingly, we observed activation of the PvMEl promoter by antioxidant compounds such as GSH, ascorbic acid, and NADPH. GSSG, expected to shift the cellular redox status toward the oxidized state and to thereby aggravate oxidative stress, re-

sulted in lower induction levels relative to GSH. The analysis of redox activation of a cytosolic SOD gene has led to similar paradoxical results, showing for severa1 antioxidants a complete lack of promoter response to oxidized forms and a strong induction by reduced forms. It has been suggested that GSH acts directly as an antioxidant and simultaneously activates the panoply of stress genes (Her- ouart et al., 1993).

An alternative explanation for the observed effects can be developed, if alterations of intracellular H,O, levels brought about by antioxidants are considered. H,O, is an important regulatory molecule (Levine et al., 1994) and is also directly involved in certain defense reactions such as lignification (Walter, 1992) and oxidative cross-linking of cell-wall proteins (Bradley et al., 1992). H,O, is removed by catalases, peroxidases, and a mechanism termed the Halli- well-Asada pathway (Fig. 4). Feeding any of the reduced forms into the pathway may lead to H,O, reduction and its removal. This may in turn activate mechanisms to replen- ish H,O,. SOD is involved and NADP-ME could be involved in mechanisms producing H,O,. Both the NADP-ME and the SOD promoter are unresponsive to exogenous H,O, (Herouart et al., 1993; this paper). H,O, has been implicated in the regulation of the salicylic acid- mediated induction of systemic acquired resistance in plants. Salicylic acid binds to and inhibits the activity of a catalase, thereby increasing cellular H,O, and inducing resistance (Chen et al., 1993). If fed to bean leaves, salicylic acid does not have an effect on PvMEl mRNA accumulation early in the treatment like GSH, but exhibits only a delayed response, which may be due to additional regulatory mechanisms. It is interesting that salicylic acid also induces oxidative cross-linking in bean, as does GSH in low concentrations (optimum 50 pm). At higher GSH concentrations (1 mM) the response is slower, presumably reflecting some H,O, destruction as a competing reaction (Bradley et al., 1992). Hence, plants seem to react very sensitively to alterations in cellular H,O, levels and redox perturbations or to compounds affecting these levels. In this context, the gene for cytosolic ME, whose enzyme protein produces NADPH, may be another “dancer to a redox tune” (Garcia-Olmedo et al., 1994).

ACKNOWLEDGMENTS

We thank Bianca Sporl and Jutta Babo for excellent technical assistance, Wolfgang Staiber for help with the artwork, Seid F. Anvari for help with tissue embedding, and Christopher J. Lamb for critically reading the manuscript. We are grateful to Mike Bevan for providing pBI 101.4 and to Christopher J. Lamb and co-workers for supplying funga1 elicitor.

Received October 24, 1994; accepted April 8, 1995. Copyright Clearance Center: 0032-0889/95/ 108/0949/12

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