Antisense DNA for an ozone-inducible 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 220.127.116.11; LE-ACS6) from tomato, under the control of the cauliflower mosaic virus 35S promoter, was introduced into tobacco to generate transgenic plants. Lower rates of ozone-induced ethylene production were observed in three of seven transgenic plants than in the wild-type plants. Ozone-induced visible damage was attenuated in these three lines, and the extent of damage was positively related to the level of ozone-induced ethylene production. In the most ozone-resistant line, ozone-induced accumulation of ACC and levels of transcripts for ozone-inducible endogenous ACC synthases were suppressed compared with those in wild-type plants, demonstrating that ozone-inducible ACC synthases have a key role in the expression of leaf damage by ozone exposure. No significant differences in growth and morphology were observed between transgenic and wild-type plants. Stomatal conductance of transgenic plants during ozone exposure was higher than that of wild-type plants. These findings indicate that the introduction of antisense DNA for an ozone-inducible ACC synthase can improve the ozone tolerance of plants without reducing their gas absorption and productivity.
Ozone (O3) is an air pollutant in many industrialized and developing countries. It is the main oxidant component of photochemical smog and causes leaf damage in many plant species (Kangasjärvi et al. 1994), contributing substantially to crop loss and forest decline (Preston & Tingey 1988). Before leaf damage appears, O3 induces many biochemical reactions, such as ethylene production, reactions involved in the hypersensitive response found in pathogen infection, and reactions involved in flavonoid synthesis (Kangasjärvi et al. 1994). In the O3-induced responses, the production of ethylene, a plant hormone, is one of the earliest detectable events (Craker 1971). Tingey, Standley & Field (1976) reported that there was a correlation between the rate of O3-induced ethylene production and the extent of leaf damage. Pre-treatment of plants with inhibitors for ethylene biosynthesis, such as aminoethoxyvinylglycine and cobalt, reduces O3-induced ethylene synthesis and visible damage (Mehlhorn & Wellburn 1987; Wenzel et al. 1995; Bae et al. 1996). It has also been shown that an inhibitor of hormonal action of ethylene reduced O3-induced leaf damage (Mehlhorn & Wellburn 1987; Bae et al. 1996). An O3-sensitive cultivar of tobacco showed higher O3-induced ethylene production than that from a tolerant cultivar (Langebartels et al. 1991). These findings strongly suggest that a hormonal action of ethylene participates in the promotion of leaf damage by O3 exposure.
Ethylene is synthesized from S-adenosyl-l-methionine via 1-aminocyclopropane-1-carboxylate (ACC) in higher plants, and ACC synthase (ACS; EC 18.104.22.168) often catalyses the rate-limiting step in ethylene biosynthesis (Yang & Hoffman 1984). The genes encoding ACS comprise a divergent multigene family and are differentially regulated in expression, depending on developmental phase and environmental conditions (Kende 1993). In some solanaceous plants, mRNAs for two types of isogenes of ACS [one transiently induced within 2 h of O3 exposure (early O3-inducible ACSs), the other after a further 1 to 2 h delay in induction (late O3-inducible ACSs)] sequentially accumulate during O3 exposure (Schlagnhaufer et al. 1997, Nakajima et al. 2001). In tomato plants, 1 h of O3 exposure was sufficient to induce leaf damage, which suggests that the early O3-inducible ACSs probably have a pivotal role in the appearance of leaf damage by O3 treatment (Bae et al. 1996). In this study, we tried to suppress the expression of O3-inducible ACSs in tobacco by introducing an antisense DNA for a tomato early O3-inducible ACS and analysed the transgenic plants under O3 exposure.
Materials and methods
Construction of antisense DNA and transformation of tobacco plants
A blunt-ended cDNA for O3-inducible tomato ACS (LE-ACS6, DDBJ accession no. U74461) was ligated in an antisense orientation to the cauliflower mosaic virus 35S promoter, and replaced the β-glucuronidase coding region of the binary vector pBI121 (Fig. 1A). Tobacco plants (Nicotiana tabacum L. cv. SR-1) were then transformed by using Agrobacterium-mediated transformation (Horsch et al. 1985). Kanamycin-resistant, putative transgenic shoots were regenerated, planted in soil, and grown in the greenhouse. The transgenic tobacco lines were self-pollinated, and kanamycin-resistant T1 and then T2 lines were obtained. All experiments were performed with T2 plants that were homozygous for the transgene.
Ozone exposure analysis
Seeds of tobacco were sown in culture soil (Kureha Chemical Industry Co., Tokyo, Japan), and seedlings were grown in a controlled-environment greenhouse at 25/20 °C day/night temperatures with 70% relative humidity under a 14 h light : 10 h dark cycle. The plants were fertilized weekly with Hyponex (0·1% v/v; Hyponex Japan Licensee, Tokyo, Japan), and pots were watered daily. Seedlings that were 5 to 6 weeks old were exposed to 0·2 p.p.m. O3 in a growth chamber (230 cm × 190 cm × 170 cm) at 25 °C and 70% relative humidity, under light from metal halide lamps with a photosynthetic photon flux density (PPFD) of 300 µE m−2 s−1. Ozone was generated from an O3 generator (Sumitomo Seika Chemicals Co., Tokyo, Japan).
The rate of ethylene production was measured as described by Bae et al. (1996). Tobacco plants at 5 to 6 weeks old were exposed to 0·2 p.p.m. O3. At sampling time, leaf discs were excised and incubated in sealed flasks under light for 1 h. Then, 1 mL of gas was withdrawn from each flask, and ethylene was analysed by gas chromatograph equipped with a flame ionization detector (GC-7 A; Shimadzu, Tokyo, Japan).
For the measurement of leaf injury, plants exposed to O3 for 6 h were transferred to the control chamber, with the light remaining on. Then 24 h later, the sixth expanded leaves from the top of the plants that exhibited the most severe damage were excised from each plant and scanned with a scanner (GT7600U; Epson, Tokyo, Japan) into a computer. The area of visible damage on the leaves was calculated by using image analysis software (NIH Image; National Institutes of Health, Washington, DC, USA). The operating quantum efficiency of photosystem II (PSII) photochemistry in actinic light and diffusion resistance were measured with three independent sixth expanded leaves from the top of the plants. Measurements were performed under light (PPFD, 300 µE m−2 s−1) in the ozone exposing-growth chamber. Data were collected by using a portable fluorometer (PAM2000; H. Walz, Effeltrich, Germany) and a diffusion porometer (Super porometer, LI-1600; LiCor, Lincoln, NE, USA) according to the supplier’s manual. The data for diffusion resistance were then processed into data of stomatal conductance.
ACC was extracted and determined as described by Langebartels et al. (1991). About 500 mg of leaf samples was extracted four times with 1 mL 80% (v/v) ethanol. Extracts were concentrated to dryness, dissolved in 1 mL water, and extracted with 1 mL chloroform. The upper phase was concentrated to dryness and dissolved in 1 mL water. The amount of ACC was determined according to Lizada & Yang (1979).
RNA extraction and Northern blot analysis
Northern blot analysis was used to confirm the expression of antisense DNA in transgenic plants. Total RNA from wild-type (SR-1) and transgenic tobacco leaves was extracted with sodium dodecyl sulphate (SDS) and phenol as described by Nakajima et al. (1995). Five micrograms of total RNA was separated by electrophoresis in a 1·2% agarose gel that contained 1·8% of formaldehyde and then transferred to a nylon membrane (Hybond N+ Amersham Pharmacia Biotech, Tokyo, Japan). Pre-hybridization and hybridization were performed as described by Nakajima et al. (1995). The probe was prepared by using a multiprime labelling system (Amersham Pharmacia Biotech) with [32P]dCTP (3000 Ci mmol−1) and full-length LE-ACS6 cDNA. The filter was washed twice with 2 × SSPE [0·02 m NaH2PO4 (pH 7·4), 0·3 m NaCl, 2 mm ethylenediaminetetraacetic acid] and 0·1% SDS for 20 min at room temperature and twice with 2 × SSPE and 0·1% SDS for 30 min at 42 °C. The filter was exposed to a Bio-Imaging plate (Fuji Film Co., Tokyo, Japan), and the autoradiograph was analysed with a radioisotope imaging analyser (BAS2000; Fuji Film Co.).
cDNA cloning for O3-inducible ACSs in tobacco
Complementary DNAs for O3-inducible ACSs from tobacco leaves were cloned by reverse transcription (RT)-polymerase chain reaction (PCR). Total RNA (0·4 µg) from O3-treated leaves was used for reverse transcription at 42 °C for 30 min using RT-PCR beads (Ready-To-Go; Amersham Pharmacia Biotech). The cDNA was amplified by PCR with specific primers. The primers were made on the basis of conserved amino acid sequences of tomato ACSs; their sequences were TT(TC)CA(AG)GA(TC) TA(TC)CA(TC)GGI(TC)TICC and GTICCIA(AG)IGG (AG)TTIGAIGG(AG)TT. The PCR conditions were 1 min at 94 °C for denaturation, 2 min at 55 °C for primer annealing, 3 min at 72 °C for primer extension, for 50 cycles. The amplified cDNAs were subcloned into a pGEM-T Easy kit (Promega, Madison, WI, USA), and 40 clones related to ACS were sequenced with an ALFred sequencer (Amersham Pharmacia Biotech). They were classified into two types of O3-inducible ACSs (NT-ACS2 and NT-ACS6). For RNase protection assays, these cDNAs were subcloned into pBluescript SK(–) (Stratagene, La Jolla, CA, USA). One plasmid harbouring NT-ACS2 was named pNTACS2; another harbouring NT-ACS6 was named pNTACS6.
RNase protection assay
An RNase protection assay was performed according to Nakajima et al. (2001) with an RNase protection kit (Boehringer, Mannheim, Germany). pNTACS2 and pNTACS6 were linearized with BsrGI and EcoRI, respectively. Labelled RNA probes were prepared by in vitro transcription with T3 RNA polymerase and 1·85 MBq of [32P]CTP, and then the probes were purified and extracted from 4% polyacrylamide–urea gel. Hybridization was carried out with 10 µg of total RNA and 32P-labelled RNA probe (800 Bq) at 45 °C for 16 h. After the hybridization was terminated, RNase treatment and proteinase treatment were carried out. Undigested fragments were purified and precipitated by phenol/chloroform extraction and ethanol precipitation, dissolved in 5 µL of sterilized water, and mixed with 7 µL of loading buffer. Heat-denatured samples were separated on 4% polyacrylamide–urea gels and analysed with the radioisotope imaging analyser.
Generation of transgenic plants
Transgenic tobacco plants were generated with an antisense DNA for an early O3-inducible tomato ACS (LE-ACS6). Seven transgenic lines that showed kanamycin resistance (a marker of successful transformation) were obtained. Northern blot analysis using a cDNA for LE-ACS6 as a probe showed constitutive expression of the antisense DNA in all these transgenic lines, but not in the control, non-transgenic plants (data not shown). The transgenic plants did not differ noticeably from the control plants in terms of germination, growth, development, or appearance under normal conditions.
O3-induced ethylene production
To elucidate the effectiveness of antisense LE-ACS6 in suppressing O3-induced ethylene production, we measured the rate of ethylene evolution from transgenic and control plants during exposure to 0·2 p.p.m. O3. In control plants, ethylene production was observed as early as 1 h after the start of the O3 exposure, increased up to 4 h, and then decreased (Fig. 1B). In contrast, in three transgenic lines (AsACS1, AsACS2, AsACS3), ethylene production was hardly apparent at 1 h. Although the peak of ethylene production was observed at 4 h also in these transgenic plants, the levels were markedly lower than those of control plants. The most remarkable line was AsACS1, whose ethylene production was about one-third that of control plants.
O3-induced leaf damage
When the control plants were exposed to 0·2 p.p.m. O3 for 6 h, their leaves withered, and necrosis appeared on the surface 24 h after the end of exposure. The injury tended to be more extensive in older leaves than in younger leaves (Fig. 2A). In comparison, the three transgenic lines with lower ethylene production showed less visible damage than the control plants, line AsACS1 showing the highest resistance to the O3 treatment (Fig. 2A & B). The extent of visible damage of AsACS1 plants was approximately one-half or one-third that of control plants.
In control plants, the operating quantum efficiency of PSII photochemistry in actinic light at 6 h O3 exposure declined to 69% of the initial value. In transgenic plants, it declined more gradually, that of AsACS1 being the most resistant (Fig. 3). After 6 h O3 exposure the efficiency was 90, 78 and 82% of the initial value in AsACS1, AsACS2, and AsACS3, respectively. AsACS1 was therefore used for further biochemical analyses.
O3-induced accumulation of ACC and transcripts of endogenous ACS genes of tobacco
When we attempted to measure the ACS activity of O3-exposed tobacco leaves, the activity was too low to be determined accurately by the conventional method (Lizada & Yang 1979) and therefore ACC content was measured instead. In control plants, the amount of ACC was increased by O3 exposure, reaching a peak 4 h after the start of the O3 exposure (Fig. 4); the pattern of induction resembled that of ethylene synthesis (Fig. 1B). By contrast, the ACC content did not increase at all during the O3 exposure in the transgenic plants (AsACS1) (Fig. 4).
To investigate whether the expression of endogenous O3-inducible ACS genes, if any, of tobacco is suppressed in transgenic plants, we first tried to identify tobacco genes encoding O3-inducible ACSs. By screening a cDNA library with RT-PCR, we isolated two kinds of cDNAs for ACSs from O3-exposed tobacco. The nucleotide sequence of one of them was identical to that of NT-ACS2 (GenBank accession no. aj005002), and the other cDNA encoded a novel ACS with 73% similarity to NT-ACS1 (GenBank accession no. x65982). We named it NT-ACS6 (GenBank accession no. af392978). Phylogenetic analysis comparing these cDNA fragments to tomato O3-inducible ACS genes revealed that NT-ACS2 is similar to LE-ACS1A and NT-ACS6 is similar to LE-ACS6. We then investigated the accumulation pattern of their transcripts during O3 exposure with their antisense RNA probes. An RNase protection assay showed that the expression of both NT-ACS2 and NT-ACS6 are O3-inducible (Fig. 5). The level of transcript for NT-ACS2 increased at 1 h of O3 exposure and was maintained at high levels until 6 h. The accumulation of transcript for NT-ACS6 was transient and reached a peak at 2 h. Accumulation of these transcripts was suppressed in the transgenic plants (AsACS1), compared with that in control plants (Fig. 6).
Air pollutants are absorbed into plant tissues mainly through the leaf stomata, and the size of stomatal aperture is an important factor in the resistance of plants (Kondo & Sugahara 1978). To check that the enhancement of resistance to O3 in the transgenic plants was not caused by a decrease in O3 absorption, the diffusion resistance of transgenic and control plants during O3 exposure was measured and used to calculate the stomatal conductance. The stomatal conductance of both control and transgenic plants was increased during the initial 1–2 h of O3 exposure, and then decreased. Transgenic plants showed higher stomatal conductance than the control plants by 2 h and it then declined to control levels (Fig. 7). These results suggest that the transgenic plants have a potentially higher O3 absorption ability than control plants.
By introducing antisense DNA for a tomato ACS (LE-ACS6), we generated three lines of transgenic tobacco plants that showed decreased O3-induced ethylene production and decreased leaf damage. The extent of damage of transgenic lines was positively related to the level of O3-induced ethylene production (Figs 1B, 2B & 3). This finding agrees with the hypothesis that ethylene is an important factor that determines plant sensitivity to O3 (Mehlhorn & Wellburn 1987).
We generated seven transgenic lines that showed constitutive expression of the antisense DNA, but only three lines exhibited lower O3-induced ethylene production. These three lines showed different O3-induced ethylene production rates, suggesting that the effect of introduction of the antisense DNA might be dependent on their copy numbers and/or position of the host genome.
In the most O3-resistant line, AsACS1, the O3-induced accumulation of transcripts for endogenous ACSs was suppressed (Fig. 6), and the amount of ACC was not increased by O3 exposure (Fig. 4). The ACC content rather decreased slightly during O3 exposure in the transgenic plants. This is probably because ACC content reflects both its production via ACS and its consumption via ACC oxidase, and only the former is suppressed in the transgenic plants, whereas both of these reactions (enzymes) are induced by O3 (Nakajima et al. 2001).
Higher stomatal conductance was observed in the transgenic plants than in the control plants during O3 exposure (Fig. 7). Naturally ozone at this level closes the stomata and interestingly they close in both lines at about the same rate, indicating that visible injury is not directly related to stomatal closure. Therefore the introduction of the antisense ACS gene improved the O3 resistance of the host plants not by affecting the incorporation of O3, but by reducing ethylene-induced reactions that are detrimental to the plants.
As mentioned in the Introduction, O3 induces the expression of two types of ACSs in tomato and potato leaves. The expression of one of them, the early O3-inducible type, is transiently induced within 2 h after the start of O3 exposure. The other, the late-O3-inducible type, is gradually induced with a further 1 to 2 h lag (Schlagnhaufer et al. 1997, Nakajima et al. 2001). To identify the O3-inducible ACSs in tobacco leaves, we isolated two kinds of cDNAs for O3-inducible ACSs from tobacco leaves, NT-ACS2 and NT-ACS6. Their nucleotide sequences had high homology to those for tomato early O3-inducible ACSs, and the accumulation patterns of their transcripts (Fig. 5) resembled those of early O3-inducible ACSs in tomato (Nakajima et al. 2001). These results suggest that NT-ACS2 and NT-ACS6 have similar physiological roles to those of early O3-inducible ACSs in tomato. In tomato and Arabidopsis thaliana (L.) Heynh., the expression of early O3-inducible ACSs was also induced by other environmental stimuli, such as touching, wounding, salinity, and heavy metals (Oetiker et al. 1997; Nakatsuka et al. 1998; Arteca & Arteca 1999; Tatsuki & Mori 1999; Nakajima et al. 2001). NT-ACS2, which was identified as an O3-inducible ACS in this study, also has been reported as a multistress-responsive ACS in tobacco leaves (Ge et al. 2000). Therefore, this type of ACS appears to contribute to ethylene synthesis in response to a variety of stress factors in many plant species.
Our previous report indicated that 1 h exposure to O3 was sufficient to induce damage in tomato leaves (Bae et al. 1996). This suggests that reactions that cause leaf damage can be triggered within 1 h after the start of O3 exposure. The results of the present study indicate that the suppression in expression of both NT-ACS2 and NT-ACS6 decreased O3-induced ethylene production and improved O3 resistance, indicating that these O3-inducible ACSs strongly contribute to the regulation of O3-inducible ethylene production and trigger reactions to leaf damage by O3 exposure.
The antisense DNA effectively suppressed the O3-induced induction in the expression of two endogenous tobacco ACS genes, NT-ACS2 and NT-ACS6, although heterologous tomato DNA was used as the antisense DNA. However, the nucleotide sequence homology between LE-ACS6 and the isolated cDNAs encoding the two tobacco ACSs was high, 72·8% with NT-ACS2 and 90·2% with NT-ACS6, and heterologous antisense DNAs with similar homologies have been successfully used to inhibit the expression of other plant genes (Salehuzzaman, Jacobsen & Visser 1993; Bejarano & Lichtenstein 1994). Therefore, manipulation of antisense DNA appears to be an effective technique for suppressing the expression of plant genes when the nucleotide sequence of antisense DNA has significant similarity to that of host genes.
We succeeded in decreasing the O3-induced ethylene production by introducing the antisense DNA for LE-ACS6, but the suppression was incomplete. Although it may be possible to obtain a thoroughly suppressed line with further production or selection of transgenic plants, those highly ethylene-suppressed lines may not survive during regeneration after Agrobacterium infection. It has been shown that the early O3-inducible ACS was also induced by an elicitor and wounding in tomato plants (Oetiker et al. 1997), probably regulating ethylene production after pathogen infection. Knoester et al. (1998) reported that the suppression of ethylene signalling by ectopic expression of mutated ethylene receptors made the plant sensitive to infection with soil-borne fungi, indicating that ethylene signalling is essential for the acquisition of some disease resistance. Therefore, highly suppressed lines might become sensitive to infection or not develop normally during the regeneration processes.
In conclusion, our results show that the constitutive expression of antisense DNA for O3-inducible ACS could improve the O3 tolerance of tobacco plants through suppressing the expression of early O3-inducible ACSs, indicating that these ACSs have key roles in the appearance of leaf damage by O3 exposure. As O3-inducible ACSs have been reported from several plant species (Schlagnhaufer et al. 1997, Tuomainen et al. 1997; Vahala, Schlagnhaufer & Pell 1998), this technology could be used for improving O3 tolerance in agriculturally and horticulturally important plant species. Moreover, because the expression of these O3-inducible ACSs has been induced also by other factors such as an elicitor or wounding (Oetiker et al. 1997; Tatsuki & Mori 1999), this technology may also be effective in conferring resistance to other biotic and abiotic stresses in plants.
This study was supported in part by the Special Coordination Fund of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.