Proc. Nati. Acad. Sci. USA Vol. 85, pp.

8805-8809, December 1988 Biochemistry

Reduction of polygalacturonase activity in tomato fruit by antisense RNA

(plant transformation/cauliflower mosaic virus 35S promoter/lycopene accumulation)

RAYMOND E. SHEEHY, MATTHEW KRAMER, AND WILLIAM R. HIATT*

Calgene, Inc., 1920 Fifth Street, Davis, CA 95616

Communicated by E. Peter Geiduschek, August 4, 1988

ABSTRACT Polygalacturonase [PG; poly(1,4-a-Dgalacturonide) glycanhydrolase; EC 3.2.1.151 is expressed in tomato only during the ripening stage of fruit development. PG becomes abundant during ripening and has a major role in cell wall degradation and fruit softening. Tomato plants were transformed to produce antisense RNA from a gene construct containing the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in reverse orientation. The construct was integrated into the tomato genome by Agrobacterium-mediated transformation. The constitutive synthesis of PG antisense RNA in transgenic plants resulted in a substantial reduction in the levels ofPG mRNA and enzymatic activity in ripening fruit. The steady-state levels of PG antisense RNA in green fruit of transgenic plants were lower than the levels of PG mRNA normally attained during ripening. However, analysis of transcription in isolated nuclei demonstrated that the antisense RNA construct was transcribed at a higher rate than the tomato PG gene(s). Analysis of fruit from transgenic plants demonstrated a reduction in PG mRNA and enzymatic activity of 7090%. The reduction in PG activity did not prevent the accumulation of the red pigment lycopene. A number of those mRNAs which increase in abundance during the ripening of tomato fruit have been cloned as cDNAs (1, 2), including the mRNA for polygalacturonase [PG; poly(1,4-a-D-galacturonide) glycanhydrolase, EC 3.2.1.15] (3-5). PG is involved in cell wall degradation and is the most extensively studied enzyme involved in ripening (6, 7). Characterizations of PG cDNAs have indicated that the polypeptide has a molecular weight of S51,000, including a 71-amino acid N-terminal sequence which may be involved in export of PG to the cell wall (3, 5). Ripening results in major changes in tomato fruit, including color development and a softening in texture (8). Color development is due to the accumulation of carotenoids, primarily the red pigment lycopene (9). PG mRNA (4, 10) and protein (11, 12) accumulate rapidly after the onset of ripening and extensive studies have demonstrated a major role for the enzyme in softening (13-15). In addition, the possibility that PG regulates other aspects of fruit ripening has been suggested (16). Slow and nonripening mutants of tomato have been described whose pleiotropic effects on ripening include a reduced synthesis of lycopene (8). These mutants are also deficient in the synthesis of PG mRNA (10) and protein (15). The use of antisense RNA to specifically inhibit PG gene expression provides an alternative to genetic analysis for investigating the function of PG during fruit ripening. Antisense RNA has been shown to regulate gene expression in bacteria (17), Dictyostelium (18), Drosophila (19), Xenopus oocytes (20), and mammalian cells (21, 22). Antisense RNA has been used in plants to inhibit transient expression in
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

electroporated protoplasts (23) and to reduce the expression of an Agrobacterium gene in transformed plants (24). The constitutive expression of an antisense chalcone synthase gene has been shown to alter flower pigmentation in transgenic petunia and tobacco (25). Here, antisense RNA is used to inhibit gene expression during fruit development. The construction, introduction into tomato plants, and subsequent transcription of a PG antisense gene is described. The expression of the PG antisense construct resulted in a substantial reduction in the levels of PG mRNA and enzymatic activity during fruit ripening. Lycopene accumulation was not prevented in fruit with reduced PG activity so that color development characteristic of red ripe fruit occurred.

MATERIALS AND METHODS

PG Antisense Binary Plasmid Construction. The structure of binary plasmid pCGN1416 is shown in Fig. 1. The multiple cloning site of pUC-13 cm (26) was replaced with a synthetic polylinker containing sites for the restriction enzymes EcoRI, Sal I, Bgl II, Pst I, Xho I, BamHI, and HindIll to produce pCGN786. pCGN783 (27) was digested with HindIll and BamHI to produce a fragment containing the cauliflower mosaic virus 35S promoter, the TnS neomycin phosphotransferase gene, and the transcript 7 3' termination region. This fragment was ligated to HindlI- and Bgl II-digested pCGN786. The resulting plasmid was digested with Sma I to remove the neomycin phosphotransferase gene and then ligated to create pCGN1410. PG gene sequences were derived from full-length cDNA clone F1 (3). A 1.6-kilobase (kb) EcoRI fragment containing the entire PG open reading frame was made blunt by using the large fragment of DNA polymerase I and inserted in the reverse orientation into the Sma I site of pCGN1410 creating pCGN1414. pCGN1416 resulted from digestion of pCGN1414 with Sal I and insertion of pCGN1414 into the Sal I site of an Agrobacterium binary vector, pCGN1206. pCGN1206 was derived from the binary vector plasmid pCGNS94 (27). This vector contained the right and left borders of the transferred DNA (T-DNA) region and the neomycin phosphotransferase gene expressed from the octopine synthase promoter. pCGNS94 was digested with HindIl, made blunt with the large fragment of DNA polymerase I, and ligated to a Sal I linker (GGTCCACC) to create

pCGN1206.
Plant Transformation and Materials. Tomato cotyledons (Lycopersicon esculentum cv. UC82B) were transformed by co-cultivation with Agrobacterium tumefaciens harboring pCGN1416 as described by Fillatti et al. (28). Transformed plants were selected by growth on kanamycin and screened by assaying leaf tissue for neomycin phosphotransferase activity (29). Isolation of Nuclei and in Vitro Transcription. Nuclei were isolated from tomato fruit according to method II of Luthe
Abbreviation: PG, polygalacturonase. *To whom reprint requests should be addressed.

8805

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Biochemistry: Sheehy et al.

pCGN1 410

Proc. Natl. Acad. Sci. USA 85 (1988)

pUC cm
35S 5' Tr 7 3'

Sal I
pCGN1414
I

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-

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FIG. 1. Construction of the binary plasmid pCGN1416. LB, left border of the transferred DNA (T-DNA) region of the Ti plasmid of Agrobacterium tumefaciens; gent, gentamycin-resistance gene; pUC, pUC plasmid origin of replication; cm, chloramphenicol-resistance gene; 35S 5', cauliflower mosaic virus 35S promoter; PG F1, full-length PG cDNA (base pairs 1624 to 1) in the antisense orientation; Tr 7 3', 3' region of transcript 7 of the Ti plasmid; Ocs 3', octopine synthase 3' region; npt II, kanamycin-resistance gene; Ocs 5', octopine synthase 5' region; RB, right border of the T-DNA region of the Ti plasmid; pRK2, pRK290 broad host-range replicon.

and Quatrano (30) except the homogenate was applied to two consecutive discontinuous gradients of 5 ml of 80% (wt/vol) Percoll (Pharmacia) on 5 ml of 2 M sucrose. Isolated nuclei were used for in vitro transcription and RNA was isolated as described (31), except the ethanol precipitate was resuspended in 10 mM Tris'HCI, pH 7.5/1 mM EDTA and applied to a Sephadex G-25 prespun column (Boehringer Mannheim). Isolation of total and poly(A)+ RNA from leaf and fruit tissue was performed as described (3) except that polysaccharides were removed by batch adsorption to cellulose (Sigmacell 50, Sigma) in 20 mM Tris'HCl, pH 7.5/0.5 M NaCI/1 mM EDTA/0.1% NaDodSO4. RNA Hybridizations. RNA gel blotting and the synthesis of 32P-labeled probes was performed as described (3) with the addition of poly(A)+ RNA at 10 ug/ml (Boehringer Mannheim), polyanetholesulfonic acid at 500 ,ug/ml, and yeast tRNA at 200 ,g/ml to the hybridization buffer. RNA synthesized by isolated nuclei was hybridized to filters bearing unlabeled RNA synthesized from Bluescribe vectors (Stratagene Cloning Systems, San Diego, CA) as described by the vendor. Polygalacturonase Activity. Fruit pericarp tissue was homogenized with a Brinkmann Polytron in 3 vol of 1.3 M NaCl/0.05 M sodium phosphate, pH 6.0/40 mM 2mercaptoethanol. The supernatant was recovered after centrifugation (11,000 x g, 30 min) and assayed for PG activity as described (6). Lycopene Determination. A 1-g aliquot of the pellets resulting from the centrifugation step described above was extracted overnight at -20C with 20 ml of acetone/hexane, 4:5 (vol/vol). The lycopene content was determined as the absorbance at 504 nm. RESULTS PG Antisense RNA in Transformed Plants. pCGN1416 contains the full-length PG cDNA clone F1 (3) placed adjacent to the cauliflower mosaic virus 35S promoter for expression in the antisense orientation (Fig. 1). Thus, pCGN1416 should be capable of high-level, constitutive expression of an antisense RNA of -1.8 kb, if the sequence which conforms to a consensus polyadenylylation site at base positions 141 to 136 of PG cDNA F1 (3) is utilized, or 2.1 kb, if the polyadenylylation signal in the 3' region of transcript 7 is recognized. Ten independent pCGN1416-transformed plants, selected as positive for neomycin phosphotransferase activity, were screened for expression of the PG antisense construct. RNA gel blot hybridization was performed on total RNA isolated from immature green fruit. PG mRNA is absent at that stage of fruit development (3). Transcripts of 1.8 and 2.1 kb were observed in addition to a 3.6-kb RNA in 9 of the 10 transformants analyzed (Fig. 2). The 3.6-kb RNA may have resulted from improper termination at the transcript 7 pol-

yadenylylation site (32). PG antisense RNA was not detected in green fruit from transformant 1416-38 or nontransformed UC82B plants. Antisense RNA and PG mRNA Levels During Ripening of 1416-1. Transformant 1416-1 was selected as representative for expression of the PG antisense construct and analyzed in further detail. To verify the identity of the antisense transcripts, poly(A)+ RNA was prepared from leaves of 1416-1 and UC82B and hybridized with a probe specific for PG antisense RNA (Fig. 3, lanes A-C). A pattern of transcripts similar to that observed in green fruit was present only in 1416-1 (Fig. 3, lane A) and not in leaves from nontransformed UC82B plants (Fig. 3, lanes B and C). The levels of antisense RNA in fruit of 1416-1 were then compared to the normal levels of PG mRNA attained during fruit ripening (Fig. 3, lanes D-I). Total RNA was prepared from green fruit and fruit harvested 2 and 6 days after the onset of ripening. RNA gel blots were probed with a nick-translated PG cDNA to detect both PG sense and antisense RNA. As shown in Fig. 3, lanes D-F, the 2.1- and 1.8-kb PG antisense RNA species were present in 1416-1 green fruit RNA and appeared to persist into ripening. The level of PG mRNA was reduced in ripening 1416-1 fruit to a level that was undetectable under the exposure conditions shown in Fig. 3, lane E (see Fig. 4, below). PG mRNA was not detected in green fruit of UC82B
4

30

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FIG. 2. Analysis of pCGN1416 transformants for antisense RNA. Three immature green fruit were harvested from transformed or control plants; the tissue was pooled and used for the preparation of total RNA. The amount of RNA loaded on each lane (50 ug) was determined spectrophotometrically and further standardized by staining intensity on agarose gels. RNA gel blots were probed with a nick-translated 1.6-kb PG cDNA F1 fragment. Individual pCGN1416 transformants are designated by number as indicated above the lanes. UC, RNA from nontransformed UC82B control plants. The far-left lane contains molecular size markers (in kb) derived from a HindIII-digest of A phage DNA.

Biochemistry: Sheehy et al.

A B (C
-

Proc. Natl. Acad. Sci. USA 85 (1988)

(; H I

8807

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FIG. 3. Analysis of antisense RNA levels in 1416-1. Poly(A)+ RNA was prepared from leaves of 1416-1 (lane A) or UC82B (lanes B and C) and 20 .g of each preparation was analyzed by RNA gel blots. Lanes A-C were hybridized to a PG antisense RNA strandspecific RNA probe produced from the 5' EcoRI-BamHI fragment of PG cDNA F1 in a Bluescribe vector. Filters were treated with RNase A after hybridization to eliminate nonspecific binding. Total RNA was prepared from fruit of 1416-1 (lanes D-F) or UC82B (lanes G-I) harvested at the following times relative to the onset of ripening. Lanes: D and G, green; E and H, 2 days after onset; F and I, 6 days after onset. RNA gel blot hybridization was performed on 50 ,ug of each preparation and lanes D-I were probed with a nicktranslated 1.6-kb PG cDNA fragment. Molecular size markers (in kb) in the unlabeled lanes were derived from a HindIII-digest of A DNA.

(Fig. 3, lane G) and subsequently accumulated as a single RNA species of 1.9-kb during ripening (Fig. 3, lanes H and I). The steady-state levels of antisense RNA in fruit of 1416-1 were lower than the amount of PG mRNA which accumulated in ripening fruit from control UC82B plants. The level of PG antisense RNA in 1416-1 fruit may actually decrease later in ripening if PG mRNA contributed to the signal shown in Fig. 3, lane F. The reduction in PG mRNA levels during ripening of 1416-1 fruit was confirmed by use of a PG mRNA strandspecific probe (Fig. 4). The presence of PG mRNA was not observed in green fruit from either 1416-1 (Fig. 4, lane A) or UC82B (Fig. 4, lane D). The steady-state level of PG mRNA in ripening fruit of 1416-1 (Fig. 4, lanes B and C) was reduced by 90%o of the level in UC82B fruit (Fig. 4, lanes E and F), as determined by direct radiographic scans of filters used for
.\

1)

F'

autoradiography. Analysis of RNA from 1416-1 fruit harvested at extremely late stages of ripening (12 days after onset, data not shown) demonstrated that reduction in PG mRNA was maintained late into the ripening process. Transcription Rates of Antisense RNA. Nuclear run-off transcription was performed to determine the relative rates of PG sense and antisense transcription. Unlabeled sense (+) and antisense (-) RNA was synthesized in vitro from PG cDNA clone F1 and applied to filters for hybridization to PG antisense RNA and PG mRNA, respectively. The antisense (-) strand ofanother tomato cDNA clone (LeEF-1) was used as an internal control to allow comparison of results with different preparations of nuclei at each stage of fruit ripening. LeEF-1 RNA accumulates to -0.3% of the total mRNA in mature green fruit and then decreases during fruit ripening (C. K. Shewmaker, personal communication). Nuclei were prepared from green fruit of transformant 1416-1 and from 1416-1 and UC82B fruit 4 days after the onset of ripening and used for synthesis of 32P-labeled RNA. Nuclei from 1416-1 green fruit (Fig. 5, blot A) synthesized PG antisense RNA but not PG mRNA, as demonstrated by hybridization to only the (+) strand. A PG antisense RNA signal was not detected from nuclei prepared from fruit of control plants (Fig. 5, blot C). The level of transcription of the endogenous PG gene(s) in ripening fruit (Fig. 5, blots B and C), as determined by hybridization to the PG (-) strand, was less than that of LeEF-1, and much lower than the rate of transcription of the PG antisense construct (Fig. 5, blot B). However, the level of endogenous PG gene transcription appeared similar in transformed and control fruit. These results indicate that the level ofPG antisense transcription may be sufficiently high relative to that of the PG gene(s) to affect the PG mRNA level available for translation. Effect of Antisense RNA on Polygalacturonase Activity and Lycopene Levels. Extracts were prepared from fruit of 1416-1 and UC82B harvested at intervals during fruit ripening and the levels of PG enzymatic activity were compared (Fig. 6A). PG activity in 1416-1 fruit was reduced by -80%o throughout ripening. An analysis of ripe fruit from additional pCGN1416 transformants (Table 1) also demonstrated significant decreases in PG activity in plants expressing the antisense RNA construct. The level of inhibition observed varied from 69% (1416-7) to 93% (1416-30) in transformants with levels of antisense RNA detectable by RNA gel blots (Fig. 2). Transformant 1416-38 which was positive for neomycin phosphoA

+
PG

gin
a

LeEF-l
FIG. 5. Comparison of antisense PG and PG transcription rates in isolated nuclei. PG cDNA F1 was inserted in both orientations into a Bluescribe vector (Stratagene) and transcribed with T7 RNA polymerase to produce the sense (+) and antisense (-) strands of PG RNA. LeEF-1 antisense (-) strand RNA was produced by using T3 polymerase. RNA concentrations were determined spectrophotometrically and -2 j.g of RNA was applied to filters as indicated. Blots: PG+, PG sense RNA; PG-, PG antisense RNA; LeEF-1, LeEF-1 antisense RNA. Filters were hybridized with 32P-labeled transcripts synthesized by nuclei isolated from 1416-1 green fruit (blot A), 1416-1 fruit 4 days after the onset of ripening (blot B), and UC82B fruit 4 days after the onset of ripening (blot C). Approximately 3 x 10' cpm of incorporated radioactivity was added to hybridization mixtures and the exposure of autoradiographs was for -6 days at -70C.

FIG. 4. Comparison of PG mRNA levels in 1416-1 and UC82B during fruit ripening. The RNA preparations used in Fig. 3, lanes DI, were subjected to RNA gel blot analysis (50 ,ug per lane) and hybridized with a PG mRNA strand-specific RNA probe produced from the 5' EcoRI-BamHI fragment of PG cDNA F1 inserted in a Bluescribe vector. Total RNA was prepared from fruit of 1416-1 (lanes A-C) or UC82B (lanes D-F) harvested at the following times after the onset of ripening. Lanes: A and D, green; B and E, 2 days; C and F, 6 days.

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A
0.60.5-

Proc. Natl. Acad. Sci. USA 85 (1988)

Table 1. PG activity of ripe fruit from 1416 transformants and UC82B Lycopene content, PG activity, Plant Amm/g of tissue A540/A2M -1 1.4 0.08 -4 1.2 0.12 -7 1.5 0.17 -9 1.9 0.12 -27 1.4 0.08 -29 0.14 2.0 2.1 -30 0.04 1.5 -33 0.09 -38 1.3 0.63 1107 1.3 0.53 1109 0.62 1.6 1150 0.70 1.6 Red ripe fruit as determined by visual inspection were harvested from the indicated pCGN1416 transformants (plants -1 through -38) and transformed control plants (1107, 1109, and 1150). The latter plants were transformed with a binary vector containing constructs of the aroA gene (33) in place of the PG cDNA fragment. Approximately 60 g of tissue from two fruit was pooled and a 6-g aliquot was analyzed for PG activity and lycopene content.

'U~u
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50

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60

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FIG. 6. Comparison of PG enzymatic activity (A) and lycopene content (B) in ripening fruit. Transformant 1416-1 (solid bars) was vegetatively propagated and 10 plants together with 10 nontransformed UC82B control plants (hatched bars) were grown in a randomized block design. Two flowers on each plant were tagged at anthesis with preassigned harvest dates represented as time in days after pollination. Fruit were harvested at the assigned dates and assayed for PG enzyme activity and lycopene content. Results represent mean values and the associated standard error for 13-20 fruit at each time point. At 60 days after anthesis, the range of values for PG activity (A50/A280) was 0.30-0.81 in UC82B and 0.02-0.20 in 1416-1. Lycopene values (A504/g of tissue) ranged from 0.65 to 2.1 in UC82B and from 0.69 to 1.9 in 1416-1.

transferase activity but did not produce detectable levels of PG antisense RNA also did not have reduced PG activity levels. A quantitative relationship between the steady-state level of PG antisense RNA (Fig. 2) and the amount of inhibition of PG activity was not found. For example, the steady-state levels of PG antisense RNA appeared higher in fruit of 1416-29 than in fruit of 1416-9 (Fig. 2), but the PG activities in ripe fruit from either transformant were similar. A similar lack of correlation between antisense RNA levels and flower pigmentation was observed in the studies involving the inhibition of chalcone synthase (25). UC82B plants regenerated after transformation with control Ti plasmids (transformants 1107, 1109, and 1150; Table 1) produced fruit with normal levels of PG activity. As shown in Fig. 6B, lycopene accumulated in fruit of 1416-1 and UC82B in a similar manner. The lycopene values for ripe fruit from the transformants listed in Table 1 were also within the range of results observed for fruit from control UC82B plants (Fig. 6B) and mock-transformed plants (Table 1).

DISCUSSION Polygalacturonase gene expression is developmentally regulated in tomato. Expression is restricted to ripening fruit and

results in a rapid accumulation of PG mRNA to levels which can represent 1-2% of the total mRNA in red ripe fruit (10). A corresponding increase in PG enzyme has been demonstrated immunologically (12) and by enzymatic activity (11). In the experiments presented here, the constitutive synthesis of PG antisense RNA in transgenic tomato plants effectively inhibited the accumulation of PG mRNA and enzymatic activity during fruit ripening. This result demonstrates the application of antisense RNA technology to the inhibition of an endogenous plant gene at the whole plant level. The specific reduction in PG activity provides a phenocopy of PG-deficient mutants useful for defining the functions of the enzyme during fruit ripening. The analysis of 10 independent transgenic plants, selected on the basis of the presence of neomycin phosphotransferase activity, established that the expression of PG antisense RNA in green fruit (Fig. 2) was accompanied by a reduction in PG enzymatic activity in ripe fruit (Table 1). However, a quantitative relationship between steady-state levels of antisense RNA and the degree of reduction in activity was not found in that transformants with significantly different antisense RNA levels showed approximately the same reduction in enzyme levels. We tentatively suggest (i) that the steady-state levels of PG antisense RNA measured in green fruit of the analyzed transformants (Fig. 2), and the corresponding rates of transcription of the antisense constructs, may have been above a threshold level required for maximum inhibition of PG gene expression and (ii) that the primary effect is exerted during a brief period of time early after the synthesis of PG mRNA. We also note that PG antisense RNA did not totally eliminate PG mRNA and enzyme activity. This is characteristic of previous antisense RNA studies in that the inhibition oftarget gene expression is incomplete. In the experiments presented here, the steady-state levels of PG antisense RNA in green fruit were lower than the amount of PG mRNA which normally accumulates during ripening. However, a comparison of transcription rates showed much higher levels of antisense RNA synthesis from the 35S promoter relative to the expression of the endogenous PG gene(s). The fact that PG mRNA can accumulate to >1% of the total mRNA while the gene is transcribed at a relatively low rate suggests that it is the efficiency of posttranscriptional processing and stability of PG mRNA rather than the transcription rate which are responsible for its high level of accumulation. In contrast, the relatively high

Biochemistry: Sheehy et al.

transcription rate yet lower steady-state level of PG antisense RNA indicate a lower stability for PG antisense RNA than for PG mRNA. However, a high rate of antisense RNA synthesis will be present in fruit at the stage of development when PG gene transcription is initiated. A transient excess of antisense to sense RNA could then exist in the nucleus and result in a rapid and effective block in PG mRNA accumulation at the level of maturation or transport. The results presented here suggest a mechanism exerted at the posttranscriptional level since the transcription of the endogenous PG gene(s) did not appear to be significantly inhibited by the synthesis of PG antisense RNA. The ability of PG antisense RNA to inhibit PG gene expression would not have been predicted from steady-state PG antisense RNA levels and demonstrates that factors, such as transcription rate, mRNA stability, and inducibility, should be considered in determining the suitability of a target gene for inhibition by antisense RNA. The ability to regulate the expression of the PG gene has agricultural importance because of the role of the enzyme in fruit softening. A number of ripening mutations which result in fruit deficient in PG activity have been characterized (8, 15, 16). However, these mutations, which include ripening inhibitor (rin), nonripening (nor), and never ripe (Nr), have multiple effects on the ripening process. All have impaired color development and accumulate only low amounts of the major red pigment lycopene. The use of PG antisense RNA to cause a specific reduction in PG expression allowed a further evaluation of the relationship of PG activity and color development. Lycopene levels in the range that is characteristic of red ripe fruit were observed in fruit from all of the analyzed 1416-transformants (Table 1). These levels of lycopene in fruit that contain 10-20% of normal PG activity can be contrasted with levels found in the Nr mutant which has 15% of normal PG activity and accumulates only a trace of lycopene (8). Thus, the use of antisense RNA technology has allowed a separation of PG activity from lycopene accumulation in the development of tomato fruit with low levels of the enzyme and normal color development.
Note Added in Proof. While this paper was in press, Smith et al. (34) published similar results. We thank Donald Helinski, Robert Goodman, Christine Shewmaker, and Belinda Martineau for critical reading of this manuscript; Kristin Summerfelt for tomato transformation; Sharon Lafferty for technical assistance; and Lynn Bedilion for help in the preparation of this manuscript.

Proc. Natl. Acad. Sci. USA 85 (1988)

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