GSAT. ALAD. CPO. Chll leaf number. ATPase. Cytf. D1. CP29. CP1 leaf number. W T W T W T W T W T. 5. B. 11 13. W T W T W T W T W T. 5 7 9 11 13. Figure 4.
Plant Physiol. (1 997) 113: 1101-1 1 1 2
Reduction of Uroporphyrinogen Decarboxylase by Antisense RNA Expression Affects Activities of Other Enzymes lnvolved in Tetrapyrrole Biosynthesis and Leads to Light-Dependen t Necrosis' Hans-Peter Mock and Bernhard Cri"* lnstitut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, D-06466 Gatersleben, Germany pyrrole ring to form coproporphyrinogen 111 (Akthar, 1994). The reaction that is still not fully understood is the one catalyzed by UROD. Additional oxidation of coproporphyrinogen leads to protoporphyrin IX. Subsequent chelation of Mg2+ or Fe2+ directs protoporphyrin IX toward chlorophyll or heme. Chlorophyll synthesis is exclusively located in plastids, whereas heme synthesis is found both in plastids and mitochondria (Little and Jones, 1976; Smith et al., 1993; Jacobs and Jacobs, 1995). However, genes or cDNA sequences coding for heme-synthesizing enzymes that can be translocated into mitochondria have not been identified. Much attention has been focused on the biochemistry and molecular biology of the early steps and some late steps in plant tetrapyrrole biosynthesis, but less attention has been paid to the control of the porphyrin-synthesizing pathway from ALA to protoporphyrin IX. We recently published the initial characterization of the first plantspecific nucleotide sequence encoding the tobacco (Nicotiuna tabacum) and barley (Hordeum vulgare) UROD (Mock et al., 1995). Light-stimulated increases in the steady-state levels of UROD RNA and protein were observed, whereas both levels decreased under developmental control in older leaves. The light-stimulated ALA-synthesizing capacity seems to be rate-limiting for chlorophyll synthesis (Kannangara and Gough, 1979; Avissar and Moberg, 1995).Feeding ALA to etiolated leaves leads to the accumulation of excessive protochlorophyllide and, to a lesser extent, of porphyrin, Mg-protoporphyrin, or coproporphyrin. This suggests a nonlimiting metabolite flow up to protochlorophyllide, which is then reduced in a light-dependent manner to chlorophyllide. A direct approach to determine the significance of an enzyme for the control of metabolite flux in a pathway is to progressively decrease in planta the amount of the target enzyme and to assay the extent of the catalytic deficiency. Control of metabolic pathways can be dominated by a single enzyme or can be shared with other components of the pathway (Stitt, 1994). We were interested in determin-
We introduced a full-length cDNA sequence encoding tobacco (Nicotiana tabacum) uroporphyrinogen 111 decarboxylase (UROD; EC 4.1.1.37) in reverse orientation under the control of a cauliflower mosaic virus 35s promoter derivative into the tobacco genome to study the effects of deregulated UROD expression on tetrapyrrole biosynthesis. Transformants with reduced UROD activity were characterízed by stunted plant growth and necrotic leaf lesions. Antisense RNA expression caused reduced UROD protein levels and reduced activity to 45% of wild type, which was correlated with the accumulation of uroporphyrin(0gen) and with the intensity of necrotic damage. Chlorophyll levels were only slightly reduced (up to 15%), indicating that the plants sustained cellular damage from accumulating photosensitive porphyrins rather than from chlorophyll deficiency. A 16-h hght/8-h dark regime at highlight intensity stimulates the formation of leaf necrosis compared with a low-light or a 6-h high-light treatment. Transgenic plants grown at high light also showed inactivation of 5-aminolevulinate dehydratase and porphobilinogen deaminase, whereas the activity of coproporphyrinogen oxidase and the 5-aminolevulinate synthesizing capacity were not altered. We conclude that photooxidation of accumulating uroporphyrin(ogen) leads to the generation of oxygen species, which destabilizes other enzymes i n the porphyrin metabolic pathway. This porphyrin-induced necrosis resembles the induction of cell death observed during pathogenesis and air pollution.
Tetrapyrrole metabolism provides essential components such as heme and chlorophyll for many important proteins involved in light harvesting, energy transduction, signal transduction, or detoxification (for reviews, see Beale and Weinstein, 1990; Smith and Griffiths, 1993; von Wettstein et al., 1995).A sequential conversion of glutamate to ALA, the first unique compound for tetrapyrrole biosynthesis, initiates the metabolic pathway (Kannangara et al., 1994). Two molecules of ALA are condensed to generate the first monopyrrole, porphobilinogen, which is sequentially combined to a linear and subsequently to a cyclic tetrapyrrole. The first cyclic porphyrin in the pathway, uroporphyrinogen 111, is decarboxylated at the acetate side chain of each This work was partially supported by a grant from the Deutsche Forschungsgemeinschaft (no. 936 /3-1). * Corresponding author; e-mail grimm@ipk-gaterslebende; fax
Abbreviations: ALA, 5-aminolevulinate; ALAD, 5-aminolevulinate dehydratase; CPO, coproporphyrinogen 111 oxidase; PBGD, porphobilinogen deaminase; UROD, uroporphyrinogen I11 decarboxylase.
49-39482-5136. 1101
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Plant Physiol. Vol. 1 1 3 , 1997
ing the function of UROD in controlled tetrapyrrole biosynthesis. To our knowledge, no plant mutant is currently available that is affected only on the leve1 of UROD. Yeast and Escherichia coli mutants lacking UROD activity cannot grow without heme supplement, indicating that UROD is essential in microbial tetrapyrrole synthesis (Sasarman et al., 1975; Zoladek et al., 1995).The human disease porphyria cutanea tarda is characterized by deficient UROD activity; the initial accumulating uroporphyrinogen is not stable and is oxidized to uroporphyrin, which absorbs light energy and causes phototoxic skin lesions (Sweeney, 1986). The antisense RNA technique provides an approach to specifically reduce UROD expression to study its regulatory and structural significance in tetrapyrrole biosynthesis, as well as the cellular response against UROD deficiency. Antisense genes for UROD were introduced into tobacco plants, and the consequences of partially suppressed activity were analyzed.
against spinach ALAD and Arabidopsis PBGD were kindly provided by Drs. H.A.W. Schneider-Poetsch (Botanisohes Institut Universitat Koln, Cologne, Germany) and A.G. Smith (Department of Plant Sciences, University of Cambridge, Cambridge, UK), respectively. Antisera against CP29 (barley), CP1 (barley), D1 (spinach), and the y subunit of plastid ATPase (spinach) were kindly provided by Profs. D. von Wettstein (Carlsburg Laboratory, Copenhagen, Denmark), J. Trebst (Ruhruniversitat Bochum, Bochum, Germany), and H. Strotmann (Heinrich-Heine Universitat, Institute für Biochemie der Pflanzen, Diisseldorf, Germany), respectively. The antibody Cmp (Knotzel and Simpson, 1991) cross-reacts with barley Lhcb4 (=CP29) of PSII and Lhca2 of PSI. Antiserum against Cyt f was raised by the injection of commercially available spinach protein (Sigma) into rabbits.
MATERIALS A N D METHODS
Content of chlorophyll was determined in alkaline acetone extracts (Lichtenthaler, 1987). Analysis of carotenoids was essentially as described previously (Kruse et al., 1995), except that an HPLC system equipped with a photodiode array detector (model 996, Waters) was used.
Tobacco (Nicotiana tabacum var Samsun NN) plants were transformed with a UROD antisense construct (pURODAS). The full-length cDNA encoding tobacco UROD (Mock et al., 1995) was excised (EcoRV/XbaI) from the Bluescript SK+ phagemid and cloned into the SmaI/ XbaI sites of the binary vector BinAR (Hofgen and Willmitzer, 1992). Agrobacterium-mediated leaf disc transformation was performed as described previously (Kruse et al., 1995). Primary transformants and their progeny obtained by selfing were grown under greenhouse conditions in ambient light and used for initial characterization of transformants by northern and western blot analysis. Plants used for further biochemical analyses were grown in growth chambers at 300 pmol photons m - ' ~ - ~16-h , light/ 8-h dark cycles, 24"C, and 70% RH. Illumination conditions were varied to monitor the influence of light on the formation of necrotic leaf damage. Plant material was harvested 2 h after the onset of illumination, frozen in liquid nitrogen, and stored at -8OOC. UNA Analysis
Preparation of total RNA was according to the method of Chomczinsky and Sacchi (1987). For northern blot analysis, samples containing 40 pg of RNA were separated on 1% agarose-formaldehyde gels (Sambrook et al., 1989). Equal loading of samples was controlled by staining the gels with ethidium bromide. After blotting, filters were probed with cDNA fragments labeled by random priming using [32P]dCTP(GIBCO-BRL). Western Blot Analysis
Extraction of total leaf protein, SDS-PAGE, and immunoblotting were performed as described previously (Kruse et al., 1995). Antisera against tobacco glutamate 1-semialdehyde aminotransferase, UROD, CPO, and CHLI (a subunit of magnesium chelatase) were prepared in our institute by Dr. R. Manteuffel (Kruse et al., 1995). Antisera
Determination of Chlorophyll Content and Carotenoid Analysis
Determination of Heme
Extraction and HPLC analysis of heme were performed as described previously (Schneegurt and Beale, 1986), except that acidic acetone extraction was performed without prior remova1 of chlorophyll. Heme a standard was obtained by extraction of commercially available Cyt c oxidase (Sigma). Concentrations of protoheme and heme a standard solutions were determined spectrophotometrically (Weinstein and Beale, 1983). Enzyme Assays
Activities of ALAD and PBGD were assayed as described previously (Smith, 1988). CPO and UROD were assayed essentially as described by Smith and Griffiths (1993). T, progeny with necrotic phenotypes were grown for 10 weeks, and wild-type plants were grown for 8 weeks in the greenhouse. Pratein extracts for each enzyme assay were prepared from liquid nitrogen-ground leaf material (100200 mg) with ice-cold assay buffer (500 pL). Extracts were centrifuged (15 min, 14,OOOg) and pellets reextracted with the same volume of extraction buffer. Combined supernatants were purified by gel filtration on columns (Pharmacia) equilibrated with the appropriate extraction buffer. Reactions were started by adding substrate. Assay conditions were as described previously (Smith and Griffiths, 1993). Reactions were stopped by freezing tubes in liquid nitrogen, and the tubes were stored at -20°C. Formation of porphobilinogen was detected by reaction with modified Ehrlich's reagent as described previously (Smith and Griffiths, 1993). PBGD, UROD, and CPO activities were monitored by fluorescence detection of oxidized reaction products on an HPLC system as previously described (Kruse et al., 1995).Uroporphyrinogen I and coproporphyrinogen 111
Reduced Uroporphyrinogen Decarboxylase Activity were used as substrates for UROD and CPO, respectively, and were the generous gifts of Dr. Frank Schmidt (AgrEvo GmbH, Berlin, Germany). Miscellaneous
Determination of ALA and Porphobilinogen Pool Size in Leaves ALA was determined by the fluorimetric HPLC method of Miyajima et al. (1994). Accumulation of porphobilinogen in tobacco plants was assessed by thoroughly extracting liquid nitrogen-ground material (100-200 mg) in 5% acetic acid in Eppendorf tubes with the help of a motor-driven pistil. Extracts were heated for 15 min at 95°C to allow the chemical conversion of porphobilinogen to uroporphyrin (Westall, 1952). Analysis of Accumulated Porphyrins Extraction and analysis of accumulated tetrapyrroles in tobacco leaves was essentially as described previously (Kruse et al., 1995), with the following modifications. Liquid nitrogen-ground leaf material was sequentially extracted with 50 mM potassium phosphate buffer (pH 7.8), methano1:O.l M NH,OH (9:1, v/v), and acet0ne:O.l M NH,OH (9:1, v/v). Extracts were diluted with an equal volume of methanol prior to HPLC analysis. The eluent system of Lim et al. (1983) was used, which allows the resolution of porphyrin I and I11 isomers. Porphyrin extraction does not distinguish between the reduced and oxidized forms of accumulating porphyrins in plant tissue. When the ratio of these forms is unknown, we use the term porphyrin(ogen). Identification of isolated uroporphyrin(0gen) as isomer 111 in UROD antisense plants was achieved by spiking samples with authentic uroporphyrin I or 111. lncubation of Leaf Disc with Glutamate Leaf discs (8 mm in diameter) were cut with a cork borer 1 h after the onset of illumination and placed in 1 mL of 0.1 M phosphate buffer (pH 7.0) containing 5 mM levulinic acid and 500 PM Glu (including 1 p~ L-[U-'~C]GIU)in Eppendorf tubes. After a brief vacuum infiltration, the tubes were incubated at 28°C in the light for 8 h. Leaf discs were then removed, rinsed with water, and extracted in 1 N TCA/1% SDS. Synthesized ALA was quantified by HPLC with a radioactivity detector (model LB 507A with a YG150U4D cell, Berthold, Wildbad, Germany) using radiolabeled ALA standard. RESULTS Phenotype of Transgenic Plants Transformed with UROD Antisense Cenes
A full-length cDNA sequence encoding UROD from N. tabacum (Mock et al., 1995) was inserted in reverse orientation behind the cauliflower mosaic virus 35s promoter of the binary vector BinAR, as described in "Materials and Methods." Primary transformants obtained by
1103
Agrobacterium-mediated leaf disc transformation of tobacco were grown under low-light conditions (70 pmol photons m-z s -1 ). Fifty selected transgenic lines were cultivated in the greenhouse. Many transformants grew significantly slower than wild-type plants, as shown for 2 lines in Figure 1A. Young leaves showed wild-type-like pigmentation and obtained necrotic lesions with whitish or brownish desiccated tissue with age (Fig. 1B). Root formation was also negatively affected in parallel with the impaired development of stem and leaf (Fig. 1C). TI progeny of transformation lines with a visible transgenic phenotype segregated generally into individuals with a different extent of necrotic leaf damage. Phenotypes of kanamycin-resistant progeny of transformant 2 (Fig. 1D) ranged from wildtype-like to severely affected plants with reduced growth rates, photodynamic leaf injuries, and delayed flowering. For a11 transformants investigated necrotic damage correlated with a drastic reduction in total leaf area, weight, and growth in length, but not with a reduction in leaf number (data not shown). Growth parameters for plants treated under different light regimes will be explained in a follow-up study and are presented in Table IV.
Molecular Analysis of Plants Transformed with UROD Antisense Genes
Genomic Southern blot analysis of transgenic tobacco DNA indicated one to severa1 integrations of the antisense gene into the genome (data not shown). Total RNA was extracted from leaves 4, 6, and 8 (counted from the top) of wild-type tobacco and selected primary transformants, which were grown for 8 weeks in the greenhouse, and analyzed by northern blotting (Fig. 2, top). Steady-state UROD mRNA content reached a maximum during leaf development in leaf 6 and decreased subsequently in older leaves. Plants 2 and 12 had strong necrotic lesions and a drastic reduction in the steady-state leve1 of UROD mRNA in all leaves analyzed compared with control plants. RNA content in plant 12 was below the detection limit. The RNA hybridization signal of transformant 2 increased toward older leaves, reflecting putatively the increasing amount of antisense RNA. UROD transcript levels were only slightly reduced in transformants 5 and 15, which showed wildtype-like phenotypes with only a few necrotic aberrations in older leaves. In addition to RNA analysis, the amount of UROD protein in leaf extracts of transformants and control plants was also determined using an antiserum raised against purified recombinant tobacco UROD (Fig. 2, bottom). UROD content was decreased in older leaves of control plants. Transformants had reduced UROD levels relative to control plants. Antisense plants 2 and 12 contained the lowest UROD content. The amount of UROD appeared to be only slightly decreased in older leaves of transformants 5 and 15 compared with wild type. Latter transformants did not show leaf necrosis and grew only slightly slower than control plants. The steady-state levels of UROD protein and RNA were correlated in reverse ratio to the macroscopic phenotype of the transgenic lines characterized.
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Plant Physiol. Vol. 113, 1997
Figure 1. Phenotype of transgenic tobacco plants expressing antisense RNA for UROD. A, Eight-week-old primary transformants 2 (left) and 12 (middle), and wild-type plant (right). B, Top view of primary transformant 2. C, Roots from plants depicted in A. Stem was removed and soil was washed off. D, Progeny (T,) of primary transformant 2 after growing 8 weeks on soil.
Determination of UROD Enzyme Activity in UROD
Antisense Transformants
UROD activity was assessed in leaf extracts to monitor the effects of antisense RNA suppression on the capacity to decarboxylate uroporphyrinogen. UROD activities were compared in leaves of three different developmental stages of 8-week-old wild-type plants and 10-week-old T2 progeny of UROD antisense plant 2 (Fig. 3, top). The lower panel of Figure 3 shows the UROD protein levels in each
RNA
«• •
#5
#2
SNN
#12
#15
1 1
Protein
Leaf
4 6 8
4
6
4
6
I
4
6
4
6
Figure 2. Northern blot analyses (top) of the expression of UROD sense and antisense RNA and western blot analysis (bottom) of UROD protein in different leaves of wild-type tobacco and primary transformants with UROD antisense genes (2, 5, 12, and 15). Total RNA was isolated from leaves 4, 6, and 8 (counted from the top of each plant). Forty micrograms of RNA was loaded per lane and separated on 1% formaldehyde-agarose gels. Blots were probed with the fcoRI fragment of a tobacco UROD cDNA clone. For western blot analysis equal amounts of leaf protein were loaded on SDSPACE. After transfer to a nitrocellulose filter, immunodetection was performed with antiserum raised against recombinant tobacco UROD (Mock et al., 1995).
corresponding extract. UROD activity reached a maximal level in younger leaves of control plants and decreased toward older leaves (Fig. 3, bottom). The mixture of young (leaves 1-5) and middle-aged (leaves 6-9) leaves of control plants contained similar amounts of UROD, whereas UROD was decreased in old leaves. UROD activity was reduced in all transgenic leaf extracts compared with corresponding control leaves. The amount of immunoreactive UROD declined simultaneously with enzyme activity, although a strict correlation did not exist between protein level and in vitro enzyme activity. Reduced UROD activity in transgenic plants was expected to impair porphyrin flow in the metabolic pathway and to lead to the accumulation of uroporphyrin(ogen) or other intermediates. Representative of all transformants with reduced UROD activity, in transformant 2 the predominant tetrapyrrole accumulated was uroporphyrin(ogen) III (Table I), and, to a minor extent, heptacarboxylporphyrin(ogen). The content of other metabolic intermediates such as coproporphyrin(ogen) and protoporphyrin was similar in transformants and wild-type controls (data not shown). The maximal difference in uroporphyrin(ogen) content between wild-type and primary transformant 2 was more than 300-fold. The porphyrin content of primary transformant 12, a line characterized by severe necrosis, and individual plants of T, and T2 progeny of transformant 2 was increased 50- to 500-fold compared with wild type (data not shown). In summary, maximal suppression to approximately 45% of wild-type UROD activity by consti-
1105
Reduced Uroporphyrinogen Decarboxylase Activity
100
UROD
£ 80 '1 60
GSAT
Q O
40
ALAD
20
CPO
DC
0
Chll
SNN
W T Figure 3. Comparison of UROD protein and enzyme activities in leaf extracts of UROD antisense and wild-type tobacco plants. Leaves 1 to 5, 6 to 9, and 10 to 1 3 (from the top to the base) of wild-type (n = 3) and UROD antisense plants (representative T2 progeny of primary transformant 2; n = 3) were pooled and used for preparation of protein extracts as described in "Materials and Methods." Extracts were applied to gel-filtration columns equilibrated with UROD assay buffer. After elution in the same buffer, assays were started by the addition of substrate. Product formation was analyzed by the HPLC fluorescence detection system described previously (Kruse et al., 1995). Western blot analysis of UROD was performed as described in the legend of Figure 2.
tutively expressed antisense RNA could be determined in transformants, resulting in high levels of accumulating uroporphyrin(ogen) III.
leaf number
W T W T W T W T
11
5
13
B ATPase
Cytf D1 CP29 CP1
Protein Levels of Enzymes Involved in Chlorophyll Biosynthesis and Other Plastidial Proteins in UROD Antisense Plants
We investigated the consequences of reduced UROD on the steady-state levels of proteins involved in tetrapyrrole biosynthesis (Fig. 4A) and other plastidial proteins containing mostly tetrapyrrole cofactors (Fig. 4B) in the severely Table I. Uroporphyrin(ogen) content in leaf extracts of wild-type tobacco and primary transformant Extracts were prepared by sequential extraction of liquid nitrogenground-leaf material with buffer, methanol/0.1 M NH4OH (9:1), and acetone/0.1 M NH4OH (9:1). Extracts were oxidized to obtain uroporphyrin from accumulated Uroporphyrinogen, and analyzed by HPLC with fluorescence detection (Kruse et al., 1995) using the eluent system given by Lim et al. (1983). Uroporphyrin 1
Leaf No. Wild Type
Transformant 2 pmol g~' fresh wt
4
18
6 8
12
5380 4205
9
2496
10
5
677
W T
leaf number
5
W T W T W T W T
7
9
11
13
Figure 4. Influence of UROD antisense RNA expression on steadystate levels of proteins involved in chlorophyll biosynthesis (A) and several other plastidial proteins (B) in leaf extracts of primary transformant 2 (T) and wild type (W). Antisera against glutamate 1-semialdehyde aminotransferase (GSAT), ALAD, CPO, CHLI (a subunit of Mg chelatase; Chll), Lhcb4, CP29 (a constituent of the PSII antenna complex), D1 (a protein of the PSII reaction center), the y subunit of plastidial ATPase, Cyt f, and CPI (the reaction center protein of PSI) were used for western blot analysis, which was performed as described in Figure 3. Leaves were counted from the top to the base.
damaged line 2. Equal amounts of total protein extracts from leaves of different developmental stages were prepared, subjected to SDS-PAGE, and probed with monospecific polyclonal antibodies, as indicated in Figure 4. Figure 3 shows the developmentally controlled reduction of UROD protein and activity in wild-type plants. The lower steady-state UROD levels in transgenic line 2 relative to control plants are given as a reference in Figure 4A. Comparing the levels of selected enzymes involved in tetrapyr-
Mock and Grimm
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role synthesis and photosynthesis between wild-type and transformant 2, similar intensities of immunostained proteins in corresponding leaves of wild-type plants and transformants were found (Fig. 4, A and B). We conclude that impaired UROD expression does not affect the developmentally controlled protein steady-state levels of tetrapyrrole-synthesizing enzymes or the levels of constituents of the photosynthetic apparatus with heme- and chlorophyll-binding properties. lnfluence of Reduced UROD on the Activity of Other Tetrapyrrole-Synthesizing Enzymes
Accumulation of uroporphyrin(ogen) in UROD antisense plants is connected with the observed reduction of UROD protein and activity. Reduced catalytic conversion of uroporphyrinogen could also shift the steady-state levels of early precursors in the tetrapyrrolic pathway or could be counterbalanced by reduced activities of the initial steps in ALA and porphyrin formation. We next performed assays for enzymes of the early pathway. Activities of ALAD, PBGD, and CPO (Fig. 5) were analyzed from the same control and transgenic plant material used for determining UROD activity (Fig. 3). Wild-type activities of the selected enzymes had, in general, a maximal level in extracts of young leaves, which decreased in older leaves in conjunction with the amount of immunoreacting protein. CPO activities of wild-type and transformant 2 (T2 progeny) were identical in leaves of the same age (Fig. 5, right). Western blot analysis also did not reveal any difference in CPO protein level in corresponding leaves of transgenic and control plants (Fig. 5, bottom). ALAD and PGBD activities were lower in leaves of the same age in transgenic plants compared with control (Fig. 5, left and middle). ALAD activity of young leaves of transformant 2 was only 20% of control activity. Loss of PBGD activity resembled that of UROD activity in line 2. In contrast to the activity, PBGD
ALAD
CPO
1O0
-$._ E > ._
2
80
l l e a v e s 6-9
60
40
20 0SNN
#2
I____II__ western blot analysis of enzymes involved in tetrapyrrole synthesis in representative T, progeny of line 2 and wild-type plants. Preparation of protein extracts for determining ALAD (left),PBGD (middle),and CPO (right) enzyme activities is described in the legend of Figure 4 using the appropriate assay buffer. Product formation was monitored spectrophotometrically for ALAD and fluorometrically after HPLC separation for PBCD and CPO.
Figure 5 . Enzymic activities and
Plant Physiol. Vol. 1 1 3 , 1997
the ALAD and PBGD protein content was not diminished in corresponding leaves of transgenic line 2 compared with controls. To determine the ALA-synthesizing capacity of transgenic and wild-type plants, discs of leaves 5, 6, and 7 were incubated for 8 h in the light with 14C-labeled glutamate and 5 mM levulinate, an inhibitor of ALAD, in severa1 independent experiments. Wild-type tissue had a synthesizing capacity of 12.4 nmol ALA cm-' h-', whereas in line 2 it was 13.3 nmol cm-' h-l, indicating no apparent effect on the capacity of ALA-synthesizing enzymes in UROD antisense plants compared with wild type. The ALA pool size was determined using a sensitive method with a fluorescent derivative of ALA, which was separated from other fluorescent components on HPLC (Miyajima et al., 1994). Porphobilinogen content was estimated from the amount of uroporphyrin I, which was chemically formed in heat-treated acetic leaf extracts (Westall, 1952). Uroporphyrin I and 111 isomers were separated using a modified eluent system (Lim et al., 1983). Compared with wild type, no significant accumulation of ALA or porphobilinogen could be detected as a consequence of decreased activities of ALAD and PBGD in UROD antisense plants (data not shown).
Effect of Reduced UROD Enzyme on Chlorophyll and Heme Accumulation
We next quantified chlorophyll and heme in wild-type and transgenic plants to study the consequences of reduced UROD activity on the availability of tetrapyrrole end products. Leaves of a11 primary transformants and wild-type plants grown in the greenhouse showed apparently similar green pigmentation (Fig. 1). Acetone leaf extracts of the severely injured transformant 2 and wild-type plants were analyzed to monitor minor changes in chlorophyll or heme content (Table 11).The total chlorophyll content was similar in younger leaves of transformant 2 relative to wild type, but reduced in older leaves. The increasing reduction of chlorophyll content in older leaves of line 2 (17% less chlorophyll in leaf 13 than in wild type) most likely reflects the progressive formation of necrotic and senescent tissue. The chlorophyll a l b ratio was not altered in this transformant compared with corresponding wild-type leaves. The amount of noncovalently bound heme in leaves of wild-type plants and primary transformant 2 progressively decreased during leaf development, as calculated on a fresh weight basis. Compared with wild type, transgenic line 2 contained reduced heme content in the same range as determined for chlorophyll (Table 11).We predict a similar reduction of covalently bound heme c in the analyzed antisense plants. Western blot analysis of Cyt f, a major leaf Cyt of the c-type, revealed no apparent changes in apoprotein content (Fig. 4). In summary, we observed a minor reduction of both heme and chlorophyll content in UROD antisense plants compared with control, which was pronounced in older leaves and was correlated with necrotic lesions. An altered distribution of porphyrins toward heme and chlorophyll end products was
Reduced Uroporphyrinogen Decarboxylase Activity
light-harvesting process. We determined the amount of carotenoid species in UROD antisense plants versus wildtype plants to monitor consequences of increasing photosensitivity in UROD antisense plants on the accumulation of carotenoids (Table 111). Most of the carotenoid species had their maximal accumulation in leaf 7. This leaf of 8-week-old wild-type plants or the 10-week-old primary transformant 2 grown in the greenhouse reached the final size and had a high photosynthetic capacity, as indicated by measuring CO, fixation (data not shown). The content of neoxanthin and lutein appeared to be slightly reduced in transgenic line 2 and corresponded to minor changes in chlorophyll content (Table 11). Decreased p-carotene content was also detected below leaf 9. Violaxanthin, antheraxanthin, and zeaxanthin were present in similar levels in transformant and control plants. In conclusion, results obtained for chlorophyll (Table 11) and carotenoid content (Table 111), chlorophyll a / b ratio (Table 11), and the amount of proteins of the light-harvesting pigment-binding complexes (Fig. 4) gave no evidence for obvious changes in the organization or stoichiometry of the photosynthetic apparatus in antisense plants suppressed in UROD activity.
Table II. Content of chlorophyll a and b, ratio of chlorophyll a/b, and content of noncovalently bound hemes i n leaves o f wild-type tobacco and primary transformant 2 Chlorophyll a and b content were determined spectrophotometrically in alkali acetone extracts using the formula given by Lichtenthaler (1987). For quantification of hemes, acidic acetone extracts were partially purified by solvent partition and ion-exchange chromatography prior to HPLC analysis (Schneegurt and Beale, 1986). Protoheme and heme A were identified and quantified by comparison with standards. As absolute amounts of total extractable hemes varied, values for each extraction were related to leaf 7 of wild-type plants (=100%). Values represent the mean t SD of four to six independent extractions. Leaf No.
Chlorophyll a/b Content
Chlorophyll alb Ratio
Heme Content
’
Fg g- fresh wt
%
Wild type
5 7 9 11
13
589 t 79 1122 t 48 1147.t 156 1032 i 71 795 t- 50
3.41 L 0.44 3.29 rt 0.23 3.19 L 0.20 2.99 t- 0.16 2.99 t 0.16
590 t 69 954 i 78 1046 t 148 945 t 128 662 t 6
3.49 t 0.07 3.21 t 0.22 3.02 t 0.27 3.03 rt 0.16 2.96 t- 0.03
n.d.’ 100 t 11 91 t 10
69 t 8 33 t 5
Transformant 2
5 7 9 11
13
n.d.
89 t 1 1 88 t 12 53 t 7 27 t 4
Light lntensity and Period Are Essential for the Development of Necrotic Lesions and lnactivation of ALAD
The photodestructive action of tetrapyrroles is dosedependent, which has also been shown for carcinoma cell cultures used as a model system for photodynamic therapy (Iinuma et al., 1994).The generation of singlet oxygen from coproporphyrin is dose-dependent and increases linearly with increasing concentrations of coproporphyrin (Arakane et al., 1996). We examined the extent of lightdependent necrosis in UROD antisense plants. Plants vegetatively reproduced from primary transformant 2 and wild-type plants were grown for 10 weeks under a 6-h light/l8-h dark regime with 300 Fmol photons m-’ s-’ before three different light programs were selected to dis-
n.d., Not determined.
a
1107
not observed upon reducing the supply of tetrapyrrole precursors. Determination of Carotenoid Composition in UROD Antisense Plants
Carotenoids are major components of the photosynthetic apparatus, functioning mainly in excess light-energy dissipation and contributing to a considerable extent to the
Table 111. Quantitative analysis o f carotenoids in leaves of wild-type tobacco and primary transformant 2 Extracts were prepared as for chlorophyll determination (Table II) and subjected to HPLC analysis as described previously (Kruse et al., 1995). Comaounds were identified and auantified with the helD of authentic standards. Carotenoid Content Leaf No. Neo”
Violab
Anth‘
LUtd yg g-
Zeae
p-Car‘
’ fresh wt
Wild type
5 7 9 11
13
16.7 t 1.1 29.5 t 2.6 30.7 t 1.6 24.2 t- 2.1 21.8 t 1.8
26.0 46.3 42.8 29.2 20.1
t 2.9 5 1.2 t 7.7 t 5.1 t 2.1
2.1 i 0.2 2.5 t 0.1 1.8 t 0.3 1.3 rt 0.5 0.6 rt 0.1
60.4 2 97.6 2 1 01.6 2 73.5 363.6 2
9.9 4.0 6.3 7.5 6.3
1.2 2 0.3 1.3 t- 0.4 0.8 i- 0.2 0.6 i- 0.1 0.4 t- 0.1
25.1 37.5 41.5 39.1 30.1
15.1 rt 2.1 25.1 t 2.0 21.8 rt 1.4 21.7 t 2.2 21.4 2 1.6
26.6 44.4 33.1 28.3 22.9
i_
3.6 t 0.6 t 3.9 t 6.3 t 4.1
3.0 2 0.5 2.8 t 0.3 1.5 t 0.3 1.3 i 0.6 1 .o t- 0.2
52.3 85.8 76.2 63.2 52.9
4.3 t 5.0 i 3.5 rt 1.5 t 8.4
2 6.3
1.4 % 0.3 1.1 t 0.2 0.8 t 0.2 0.4 f 0.1 0.4 t 0.1
22.1 2 3.5 40.0 t- 3.9 39.1 3- 3.9 32.6 t 2.6 21.7 3- 2.3
rt
Transformant 2
5 7 9 11 13 a
Neo, Neoxanthin.
Viola, Violaxanthin.
‘Anth,
Antheraxanthin.
Lut, Lutein.
e
2 3.5 2 6.1 -C
7.9
2 7.0
Zea, Zeaxanthin.
6-Car, 6-Carotene.
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Plant Physiol. Vol. 11 3, 1997
Mock and Grimm
Table IV. Degree of necrotic leaf damage and growth of tobacco primary transformant 2 plants under various light conditions Primary transformant 2 and control tobacco plants were grown for 1O weeks under a 6-h light (300 pE)/18-h dark cycle at constant temperature (24°C). Plants were then grown for an additional 4 weeks under these conditions or under 16-h light/8-h dark cycles at 300 or 90 pE before determination of growth parameters. Plants were visually inspected for necrotic leaf damage. Percentage of necrotic plant leaves or plants represent the mean 2 SD from three independent experiments, each comprising 6 wild type and 12 transformants. Crowth parameters were determined from one of these sets. lllumination Condition Parameter
300 pE, 16 h of Light Control
Necrotic plants (%) Necrotic leaves (%) Plant height (cm) Leaf area per plant (cm') a
n.d." n.d. 23.2 t 2.2 1149 t 111
Transformant 2
70 57 15.2 628
2 10 t5 t 6.9 C 332
300 pE, 6 h of Light
90 pE, 16 h of Llght Control
Transformant 2
Control
Transformant 2
n.d. n.d.
1023 5 2 1 11.9 2 5.6 546 2 347
n.d. n.d. 13.3 t 2.2
1554 7 2 2 7.6 C 3.3 286 C 145
18.7 C 2.4 872 C 93
554 -C 41
n.d., Not detected.
tinguish between lesion-forming and nonforming growth conditions (Table IV). Under each light regime UROD antisense plants grew slower than wild-type plants and showed reduced leaf area and size. A variation in leaf and plant size of transformants is reflected by the high SD values. Plants became photodynamically susceptible during a long photoperiod at high light, as indicated by the necrotic lesions on most of the transgenic plants. Necrotic damage in a similar series of plants of line 2 was drastically reduced when plants were exposed to low-light intensity (90 photons m-'s-' for 16 h) and short-light periods (300 photons m-' s-l for 6 h). Moreover, the necrotic leaves were less severely damaged than those grown at 16-h high-light periods. We compared UROD activity and porphyrin content of wild-type plants and line 2 plants grown under three different light regimes (Fig. 6A). Severa1 plants were pooled for preparation of protein and porphyrin extracts. The UROD activity of necrotic plants grown at high light yielded approximately 45% of control activity; the nonnecrotic plants of line 2 contained 65% of control activity. Transgenic plants without lesions grown at the other two light programs also showed a remaining UROD activity of 60% relative to wild type, although the absolute activities were markedly reduced compared with plants exposed to 16-h high-light periods. As a result of lower UROD activity porphyrin accumulated in similar amounts in necrotic and subnecrotic plants that were grown under the two moderate light regimes (Fig. 6B). Transgenic plants without visible lesions at high-light conditions had remarkably lower porphyrin content and higher residual UROD activity. A 20% difference of remaining UROD activity between necrotic and nonnecrotic plants indicates a possible threshold leve1 that provokes cell death. Growth at different light programs suggests that the reduction of UROD activity is a prerequisite for the formation of necrosis, but certain light conditions are required to induce cell death. It is still speculative if necrosis results exclusively from phototoxicity of porphyrins or if it is an induced cellular response similar to programmed cell death. Because we expected that reduced specific ALAD activity was caused by photodynamic effects in UROD antisense plants (Fig. 5), protein extracts of plants grown under these
three different light regimes were analyzed for ALAD activity. Lower specific activities of ALAD were measured in UROD antisense plants compared with wild-type plants under light-stress conditions (300 pmol photons m-' s-' for 16 h; Fig. 6C). A decrease in ALAD activity was more pronounced in necrotic plants than in antisense plants without lesions, as was the measured UROD activity. The other two light conditions did not affect ALAD activity in line 2 plants. The data strengthen the conclusion that the ALAD inactivation observed in transgenic plants with accumulating porphyrins is strictly light-dependent.
DISCUSSI ON Decrease of UROD Activity by Expression of U R O D Antisense RNA 1s Accompanied by a Light-Dependent Necrotic Phenotype
We introduced UROD antisense .genes into the tobacco genome by Agrobacterium-mediated leaf disc transformation. Among the kanamycin-resistant tobacco plants, different lines showed progressively increasing necrotic leaf damage, reduced growth rates, and reduced leaf size (Fig. 1; Table IV). Growth retardation and a decrease in leaf area are most likely consequences of cellular responses to compensate for photooxidative processes. Neither bleached nor pale green plants grown in in vitro culture or in the greenhouse were observed, indicating that the slightly lower chlorophyll content cannot be attributed to the transgenic necrotic phenotype (Table 11). The maximal decrease in chlorophyll content in severely damaged plants did not exceed 15 to 17% of control. The formation and intensity of necrotic leaf damage was correlated with the decrease in UROD transcript levels (Fig. 2) and in the amount and activity of UROD protein (Figs. 2 and 3 ) . Under high-light conditions reduction of UROD activity in primary transformants and T, and T, progeny never exceeded 55% of that in wild type (Fig. 6B), suggesting that a further decline in essential UROD activity may be lethal. Reduced UROD activities in transgenic plants were already measured in premature leaves that did not show any obvious necrotic lesions.
Reduced Uroporphyrinogen Decarboxylase Activity
A
I
16 h llght, 300 pE
f
-16 h liaht, 90 ME
4.5-
r
! 3 f
2
1.0
-
0.6
-
0.0
-
OL O
2500
t
SNN
AS,"
16 h light, 3W pE
AS.NE
SNN
1
AS
'16 h Ilght, 90E, !
i 6 h Iighl, 300 pE
SNN
AS
L LI SNN
1
SNN
16 h light, 90 pE
SNN
AS
t
AS
6 h light, 300 pE
SNN
AS
Figure 6 . Specific activities of UROD (A) and ALAD (C), and accumulation of URO (B) in wild-type (SNN) and UROD antisense (AS) plants grown under different light conditions. Growth of plants was controlled as indicated in Table IV. Error bar5 denote the SD of the mean of four replicate assays. Leaf material was harvested from leaves 5 and 6 and pooled from three plants. Under light conditions favoring leaf necrosis plant material was separately harvested from transformants without (AS,") or with the necrotic phenotype (AS,NE).
The light dependence of tetrapyrrole-induced cellular damage has been established for the action of diphenylether-type herbicides and ALA feeding (Rebeiz et al., 1984, 1988). Our results presented in Figure 6 and Table IV demonstrate that the porphyrin-induced cell death could almost be prevented under low-light (90 pE) or short-light periods (6 h). Reduced UROD activity and the subsequent accumulation of uroporphyrin I11 in transformants under different light conditions (Fig. 6) did not necessarily lead to cellular destruction, but did cause a reduction in plant growth. It remains to be seen whether light-dependent cell death can be explained entirely by oxidative damage due to accumulating porphyrins or by cellular responses mediated by increasing amounts of reactive oxygen species. Along with high-light-dependent necrosis in UROD antisense plants, transgenic plants with reduced catalase activity were recently shown to develop necrotic lesions only under high-light conditions (Chamnongpol et al., 1996)
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Limiting UROD Activities Causes Phototoxic Action of Accumulating Uroporphyrin(ogen)
Excess uroporphyrin( ogen) accumulated as a result of reduced UROD activity in transgenic plants, indicating a partia1 blockage in the metabolite substrate flow at the level of UROD. Uroporphyrinogen, like any other tetrapyrrole intermediate, is a potent photosensitizer that is oxidized by light and photosensitizes the formation of reactive oxygen species, primarily singlet oxygen, presumably by transferring excess energy from porphyrins in the excited state to oxygen. Because a11 organisms produce cyclic tetrapyrroles, they potentially contain the machinery for their own destruction. The phototoxicity of UROD antisense plants indicates that alteration of the UROD levels can barely be tolerated in certain light conditions. Referring to current models of the regulation of the tetrapyrrole pathway, it is assumed that formation of protoporphyrin from ALA does not limit the synthesis of chlorophyll and heme (Beale and Weinstein, 1990). Porphyrins are transient metabolites that do not accumulate to any large extent in plant tissues. A continuous flow of the photolabile intermediates is a prerequisite of intact cell metabolism until the end products are formed and stabilized in pigment-binding proteins. Therefore, the developmental and light-controlled expression of UROD explains its correlation with adjacent enzymes to prevent elevated levels of chlorophyll precursors. A steady-state level of UROD RNA in barley showed a light-stimulated accumulation in primary leaves and a decreasing gradient from the base to the top leaves (Mock et al., 1995), reflecting most likely the increasing requirements of tetrapyrroles in greening and premature leaves. Similarly, the content of RNA and protein was found to be development-dependent in tobacco from the top to older leaves (Figs. 2 and 3). The UROD antisense plants exhibited a necrotic phenotype resembling that of tobacco plants expressing antisense RNA for CPO (Kruse et al., 1995). These plants showed necrotic leaf damage, and their CPO activity in CPO antisense plants was maximally reduced to approximately 35% that of wild-type activity. The amount of accumulated coproporphyrin(ogen) in transformants exceeded the level in wild-type plants up to 1000-fold and was positively correlated with the degree of necrotic damage (Kruse et al., 1995). UROD antisense RNA synthesis caused a similar effect in barley and tobacco plants treated with cesium chloride (Shalygo et al., 1996). Cesium chloride incubation for 8 h leads to accumulation of uroporphyrin and causes similar leaf lesions. UROD deficiencies have been described for other organisms. Yeast mutants with defects in UROD accumulated large amounts of uroporphyrin and partially decarboxylated products accompanied by a generally low content of heme (Kurlandzka et al., 1988; Zoladek et al., 1995). The inactivation of this step in porphyrin synthesis resembles the process in the human disease porphyria cutanea tarda, resulting in a similar pattern of uroporphyrin accumulated in liver cells or excreted into urine. Familial deficiency of UROD activity is caused by mutations or is induced by exogenous factors (Nordmann and Deybach, 1990).
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Mock and Grimm
Reduced U R O D Activity Affects Activity of ALAD and PBCD, but Not of CPO
Western blot analysis was performed from selected enzymes involved in tetrapyrrole synthesis to monitor possible consequences of inhibited UROD gene expression on the control of synthesis, stability, and activity of other proteins of the pathway. The amount of glutamate 1-semialdehyde aminotransferase, ALAD, CPO, and CHLI (a subunit of Mg chelatase) of transformant 2, the line with the strongest necrotic phenotype, and wild-type plants did not significantly differ in corresponding leaves (Fig. 4A). Analysis of other transformants also revealed no changes in these protein levels (data not shown). Accumulating uroporphyrins could be the reason for the altered expression or activity of preceding enzymes of tetrapyrrole synthesis. Therefore, activity of these early enzymes was determined and correlated with the corresponding protein levels. Decelerated activities of ALAD and PBGD compared with wild type were observed. in transformant 2 during leaf development, whereas CPO and the overall ALA-synthesizing activity was not significantly altered (Fig. 5; Table 11). The loss of ALAD and PBGD activities could not be explained by the reduced content of these proteins demonstrated by immunoanalysis. ALAD was inactivated when plants were exposed to long highlight periods (16 h, 300 pmol photons m-* s-'); this treatment also caused porphyrin-induced cell death. Different mechanisms have been suggested to account for the inactivation of both enzymes. At present, a feedback mechanism as a response to diminished UROD activity or accumulated uroporphyrin is highly speculative. The enzymes of the pathway may form a protein complex to ensure sequential catalysis and to prevent porphyrin accumulation and subsequent photooxidation. A reduced amount of UROD could weaken the association with other proteins in the porphyrin-synthesizing pathway, making these enzymes more accessible to photosensitization. ALAD and PBGD are more sensitive to the photodestructive effects of accumulating uroporphyrin(ogen), which includes the generation of reactive oxygen species. The photodynamic inactivation of ALAD or PBGD did not lead to measurable protein degradation, but perhaps to oxidation or the cross-linking of amino acids, which could induce structural changes. CPO might be more tolerant against porphyrin-induced protein modification. Incubation of homogenized erythrocytes with uroporphyrin in the light and, to a lesser extent, in the dark caused inactivation of ALAD and PBGD (Afonso et al., 1990,1994),which is consistent with a higher sensitivity of both enzymes toward porphyrin-generated cellular modifications. Initial in vitro experiments with purified tobacco ALAD also demonstrated light-dependent inactivation of these enzymes after incubation with uroporphyrin or coproporphyrin (H.-P. Mock, unpublished results). Loss of UROD activity in the transformants is certainly attributed to the reduction of endogenous translatable UROD RNA and, subsequently, to lower UROD content. However, we cannot rule out that UROD was also affected by photodestructive energy of accumu-
Plant Physiol. Vol. 1 1 3, 1997
lating uroporphyrin(ogen). Oxygen-related free radicals could directly inhibit UROD (Smith and Francis, 1981; Francis and Smith; 1988; Afonso et al., 1991).
Necrotic Lesions in U R O D Antisense Plants Are Probably Caused by Reactive Oxygen Species Cenerated by Photooxidized Uroporphyrin(0gen)
The formation of leaf necrosis in UROD antisense plants due to photosensitizing uroporphyrin resembles other cellular processes against invading pathogens (Mehdy, 1994), toward ozone or UV-B irradiation (Kangasjarvi et al., 1994; Green and Fluhr, 1995), disturbed mineral nutrition (Iturbeormaetxe et al., 1995), or reduced catalase activity (Chamnongpol et al., 1996).These cellular reactions involve reactive oxygen species acting as signaling or destructive molecules (Mehdy, 1994; Pahl and Baeuerle, 1994). As indicated in catalase-deficient plants, the development of symptoms similar to the hypersensitive reaction is most likely due to signaling events triggered by increased levels of reactive oxygen species. Increased levels of reactive oxygen species generated by accumulated tetrapyrroles under high-light conditions might induce a similar cascade of cellular reactions in UROD antisense plants Severa1 reports have shown that singlet oxygen is responsible for the phototoxicity of porphyrins, but other oxygen radicals are formed as well (Hopf and Whitten, 1978; Rebeiz et al., 1984).As an example of a strong analogy to the UROD antisense RNA expression in transgenic plants, photodynamic herbicides applied to green plants exert photooxidative damage by inducing accumulation of excess tetrapyrroles. These herbicides are mainly targeted to protoporphyrinogen oxidase. Simultanwus application of the tetrapyrrole precursor ALA enhances the herbicidal effects by increasing the amount of accumulated chlorophyll precursors (Rebeiz et al., 1984, 1988). A sequence of events from altered UROD activity to the necrotic phenotype resembles protoporphyrin IX accumulation by inhibition of protoporphyrinogen oxidase (Lehnen et al., 1990). Reactive oxygen species produced in plants are normally detoxified by enzymatic and chemical antioxidants present in a11 compartments, especially in photosynthetic organelles (Foyer et al., 1994). We assume that in uroporphyrin-accumulating plants these oxygen radicals cannot be efficiently detoxified under certain conditions (light period and light intensity). The final phototoxic processes could occur in the cytoplasm when accumulating uroporphyrin leaks out of the plastids and triggers a free radical chain reaction, which may induce protection against oxidative stress, but eventually reaches a point of no return and programmed cell death. The cellular response to oxidative stress, which could stimulate the antioxidative protection system and elicit cell death in our UROD antisense plants, will be analyzed in future studies. The light-dependent transition from a subnecrotic to a necrotic phenotype in UROD antisense transformants will permit studies on the mechanism induced by reactive oxygen species to affect tetrapyrrole-induced leaf necrosis.
Reduced Uroporphyrinogen Decarboxylase Activity ACKNOWLEDCMENTS We thank Dr. Rainer Hofgen (currently at the Max-PlanckInstitut fiir Pflanzenphysiologie, Golm, Germany) for an introduction to plant transformation techniques. The technical assistance of Elena Barthel and the initial characterization of other UROD antisense plants by Livio Trainotti (currently at the University of Padova, Italy) are also gratefully acknowledged. Received November 11, 1996; accepted January 7, 1997. Copyright Clearance Center: 0032-0889/97/ 113/1101/12.
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