Antisense apple ACC-oxidase RNA reduces ethylene production in transgenic tomato fruit

Plant

Science

122 (1997) 91 99

Antisense apple ACC-oxidase RNA reduces ethylene production in transgenic tomato fruit Karen

M. Bolitho,

Michael

Lay-Yee,

Michelle

L. Knighton,

Gavin

S. ROSS”

.VlW Zclrlurlcl Received

I I June 1996: revised 17 September

1996: accepted

19 September

I996

Abstract Transgenic tomato (L~wprrsicor~ r.sc.zr/rntu~~z) plants were produced which expressed antisense copies of an apple fruit ACC-oxidase RNA. In the fruit of the primary transformants, ethylene production was reduced by over 95’14 in one of the lines assessed, and to a lesser extent in the other lines. The line showing the greatest reduction in ethylene production showed a delay in the development of colour in the transgenic fruit. Northern analysis of steady-state RNA levels using strand-specific probes indicated that fruit of the low-ethylene line had very low levels of ACC-oxidase sense-RNA. and high levels of antisense RNA. In other lines. high levels of antisense RNA were not always associated with a reduction in the levels of either sense-transcript or ethylene production. The successful reduction in ethylene production confirms the usefulness of using tomato as a model system for testing specific ripening-related genes from heterologous fruit species such as apple. Copyright 80 1997 Elsevier Science Ireland Ltd. K~~wo&:

Ethylene:

ACC-oxidase:

Fruit ripening;

Tomato;

1. Introduction The genes which encode 1-aminocyclopropane1-carboxylic acid (ACC)-synthase and ACC-oxidase, two key enzymes in ethylene biosynthesis, were first cloned from tomato [1,2]. Homologues of these genes have now been cloned from a range of other fruit species including apple [3-51, ki* Corresponding author. Tel.: 8 I54101 : e-mail: [email protected] Olh7-9451/97~$17.00

Copyright

PII SOI 68-9452(96)0453’-3

+ 64 9 8493660;

!c? 1997 Elsevier

fax:

Science

+ 64 9

Ireland

Apple:

Antisense

wifruit [6] and melon [7,8]. In tomato, manipulation of ethylene production in ripening fruit has been achieved through use of antisense RNA technology to reduce the levels of sense mRNAs encoding either of these enzymes [9, IO]. The cffects of reduced ethylene production on a range of ripening parameters have been studied in depth [11,12]. Ethylene production has also been manipulated in transgenic tomatoes by expression of prokaryotic genes which reduce the levels of Sadenosylmethionine (SAM, [ 131) or ACC [ 141. Ltd. All rights

reserved

92

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Bolitho et al.

: Plam S&ww

In addition to being an important commercial crop in its own right, the tomato represents a ‘model’ fruit which can yield information directly applicable to research on a range of other fruit crops [15]. There has been a huge investment in tomato research, and this is now paying off with both the release of new commercial transgenic cultivars (eg. Calgene’s FLAVR SAVRTM) and an improved understanding of the physiology and molecular biology of a range of ripening-related enzymes. Effective ‘model’ species for molecular biologists should also represent systems where the introduction of new or altered genes from other species can assist in elucidating gene function. This introduction of heterologous genes into model species has become common in animal models such as Drosophila (eg. [16,17]), and in plants such as Alahidopsis (eg. [l&19]) and tobacco (eg. [20.21]) but is not yet common in tomato. Down-regulation of sense-gene expression by homologous, antisense RNA has been successful in a number of plant tissues including tomato fruit (eg. [9,10]). However, the antisense technique would be more powerful if constructs containing cDNAs derived from heterologous species could be used to inhibit expression of genes of interest in model systems. There have been few attempts to down-regulate sense-mRNA expression with heterologous antisense RNA (eg. [22-241) and to our knowledge there are no reports of this approach being taken in fruit. Furthermore, while tobacco transgenics expressing heterologous antisense mRNAs showed reduced enzyme activity, and reduced accumulation of the polypeptides under investigation, this decrease was not always correlated with a reduction in levels of sense mRNA [22,23]. This suggests the mechanism of heterologous antisense gene suppression may differ from that involved in homologous antisense effects [23]. Here we report that the introduction of antisense copies of an apple ACC-oxidase mRNA into tomato produces fruit lines which show reduced production of ethylene. Our experiments further demonstrate the effectiveness of the approach of using heterologous RNAs to inhibit the expression of target genes. The experiments

122 (1997) 91-99

confirm that tomato represents a fruit system where gene constructs and physiological effects of genetic manipulation can be tested, as a prelude to manipulation in plants such as apple with longer generation times.

2. Methods 2.1. Construction qf’ untisensr ACC-osidusr constructs The entire 1.2 kb insert from the apple cDNA clone pAP4 [4] was isolated following a PstI/SphI digestion of the plasmid, and was sub-cloned into the PstI/SphI site of pUC19. The cDNA was then recovered as a XhaI fragment which was inserted into the J&I site of the binary vector pGA643 [25] to produce the pGAAP4 construct (Fig. 1). Restriction and Southern analyses were used to confirm antisense orientation of the pAP4 cDNA in pGAAP4. In this construct, expression of the

pGAAP4 12.8 kbp

Fig. I. Map of the antisense construct pGAAP4, derived from the binary vector pGA643. The portion of the DNA transferred into the plant genome is between RB (right border) and LB (left border). Expression of the antisense AP4 RNA is driven by the cauliflower mosaic virus 35s promoter. Kanamycin resistance is conferred by expression of the neomycin phosphotransferase (nptI1) gene in transgenic tissue. The approximate location of primers used in PCR-based confirmation of transgenic tissue is indicated (RAKl, RAKZ. RS2, RAGl).

antisense copy mosaic virus resulting from be selected on

of AP4 is driven by the cauliflower 35 S promoter. Transgenic tissue transformation with pGAAP4 can medium containing kanamycin.

pGAAP4 DNA was transferred from E. co/i (dH5a) into Agrohucteriunz tun~e&Gvzs (strain 2760) by tri-parental mating with E. co/i (strain 2073). A. tunw$rc.icm colonies harbouring the plasmid were then used for tomato transformation [26]. All tissue culture was performed in vented petrie dishes or pots at 26°C. with a 16 h photoperiod. Tomato (L~arywsicon c~scu/e~~tu~~~ Mill cv. UC 82B) explants were prepared by cutting the base and tips of 7 day old cotyledons. and these were preincubated overnight on KCMS [26] media. Co-cultivation began with a 5--7 min immersion of explants in a log-phase subculture of transformed A. tumqf~ciens. Explants were incubated a further 24 h on KCMS before transfer to 2 Z [26] media containing carbenicillin disodium salt (500 mg;‘l; Sigma) and kanamycin ( 100 m&l: Boehringer Mannheim). Media was renewed after IO days and thereafter every 3 weeks. Shoots up to I cm long were harvested from multiplying callus and transferred to MSSV [26] media. Root development was induced with indole-3-butyric acid (IBA) at I mg,l, on a medium containing a lower concentration of kanamycin (50 mg,‘l). Over 3 tieeks, ten plantlets were divided to produce three replicate plants of each line. Plantlets having roots I --5 cm long after IO-14 days were moved from culture to covered vermiculite trays, and then to potting mix after a 3 day hardening-off period. Three un-transformed wild-type plants were also grown from seed, alongside the transgrnics. All plants were grown under natural light in a glasshouse at approximately 25°C. with a 14 h photoperiod. The growth room was scrubbed of ethylene by inclusion of PurafilTM II.

The polymerase chain reaction was used to confirm that kanamycin-resistant plants contained the pGAAP4 T-DNA. Two primer combinations

were used with genomic DNA extracted from leaf tissue [27] as a template. Reactions were performed as described [28]. Using the primers RAKl (S-GAGGCTATTCGGCTATGACT-3’: [28]) and RAK2 (5’-AATCTCGTGATGGCAGGTTG-3’; [28]), in positive lines an 804 bp fragment was expected to be amplified from within the rz~~tl1 gene. The RAG1 (5’-GCACCTACAAATGCCATCAT-3’ [28]) and RS2 primer (5’CAGAATGTCGATAGCCTCGT-3’) combination was expected to amplify a 660 bp fragment which spanned the junction between the CaMV 35 S promoter and the AP4 cDNA in the antisense orientation.

Flowers were tagged with the date of anthesis. Fruit were harvested at breaker stage and ripened at ambient temperatures in a ventilated room (ethylene < 0.01 ppm) under artificial light. Ethylene production by individual fruit was measured 7, 4, 6. 14 or 24 days after breaker. For this measurement, individual fruit were held in sealed containers for I h. after which a I ml gas sample from the chamber headspace was removed and ethylene concentration determined by gas chromatography. Fruit colour was visually assessed against a range of control fruit standards. A Minolta chromameter was used on a sub-sample of 30 fruit assessed as ‘50% red’. In these fruit, L*. (I*, h* readings ranged from 36.7. 22.0, 19.0 to 42.9. 37.6. 31.4. respectively. Three individual fruit from each time point were trozen in liquid nitrogen and stored at - WC’. pending RNA extraction.

RNA was extracted from the pericarp of tomato tissue [29]. The RNA was size-fractionated on 1.2% agarose gels containing 0.66 M formaldehyde. and transferred to Hybond N+ membranes by capillary transfer overnight in 50 mM NaOH [4]. RNA probes incorporating [“PIUTP were synthesised using T7 RNA polymerase and either the pAP4 [4] or pJX4 plasmids (both

94

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Bolitho et al. 1 Plant Science

A 1234567

Fig. 2. Confirmation that putative transgenic tissue contains DNA from the pGAAP4 construct. In positive transgenic tissue, a band of 804 bp was expected from the RAKl/RAK2 primer combination (panel A). and a band of 660 bp was expected from the RAGI. RS2 primer combination (panel B). Lanes were loaded with: 1 kb DNA standard (Lane 1). or products of polymerase chain reactions where the DNA templates were genomic DNA extracted from putative transgenic tomato leaves (Lanes 2-4, Lines Al, A2, A3 respectively). the pGAAP4 plasmid (Lane 5), wild-type genomic DNA (Lane 6) or no DNA (Lane 7).

linearised with X6aI) as templates. The pJX4 plasmid contains an 810 bp SpeI/AccI fragment of the tomato pTOM 13 cDNA [30] cloned 5’ -+ 3’ into the SpeI/AccI sites of pBluescript II SK+. These combinations of RNA polymerase and template were used to produce single-stranded probes for the detection of either antisense apple AP4-homologous RNA, or sense tomato ACC-oxidase RNA.

122 (1997) 91-99

3.2. Production of ethylene and colour development in ripening fruit

Across all transgenic lines there were no differences in the time taken for fruit to develop from anthesis to the breaker stage (data not shown). Ethylene production by ripening wild-type tomato fruit increased in the first 4 days after the breaker stage, and declined during subsequent phases of ripening (Fig. 3). Ethylene production varied considerably between transgenic lines (Table 1, Fig. 3), with the fruit from some lines producing similar amounts of ethylene to wild-type (eg. line A2), others showing a reduction by - 50% (eg. line A3), and one line showing a reduction in ethylene production of more than 95% (line Al). The development of fruit colour was used as an indicator of fruit ripening. While there was a substantial reduction in ethylene production in a number of lines, a delay in colour development was observed only in line Al. Fruit of this line took - 7 days longer to progress from breaker to 50% red, when compared to wild-type (Table 1). 3.3. Expression of sense and antisense ACC-oxidase rnRNA

Expression of sense-strand ACC-oxidase transcripts in ripening wild type tomato fruit increased after the breaker stage, to peak after a 25-

3. Results 3.1. PCR analysis

to conjirm transjbrmation

Using genomic DNA from kanamycin resistant tomato lines as templates, all PCR amplifications produced bands of the size predicted from the pGAAP4 construct (Fig. 2). This result was taken as confirmation that the leaves of all transformed plants contained the nptI1 gene, and that all plants also contained the pGAAP4 TDNA.

0

2

4

Days after

6

14

28

breaker

Fig. 3. Ethylene production during ripening in fruit of different transgenic lines. Ethylene production during the ripening period is shown for wild-type fruit (c) and transgenic lines A I (V). A2 (A) and A3 (W).

K.M. Table I Ripening Fruit

line

in tomato

fruit transformed

Ethylene production breaker) (n = 12)

Wild type Al All A6 A3 A5 A7 Al

Bolitho et ul.

with an antisense

Plmt

version

(‘% of wild type, 4 days after

I 00 92 90 49 53 32 25 3.5

further 4 days (Fig. 4A). In the transgenic line Al which showed the greatest reduction in ethylene production, levels of sense-strand ACC-oxidase RNA were also dramatically reduced (Fig. 4A). After a 24 h exposure of a northern blot hybridised with a “P-labeled probe specific for sense-strand ACC-oxidase RNA, a signal could be detected only in breaker fruit, although after a longer film exposure time the sense-strand message could be detected in all fruit (data not shown). Line Al fruit also showed high, consistent steady-state levels of the antisense RNA derived from the apple ACC-oxidase cDNA (Fig. 4B). In line A2 (showing wild-type levels of ethylene production), there were high levels of both the sense and antisense messages, while in line A3 ( - 50% of wild-type ethylene production) sense-strand RNA levels were comparable to wild-type, while the antisense transcript did not accumulate to the levels seen in the other two transgenic lines.

4. Discussion The transformation of woody species such as apple can now be achieved, albeit at often low efficiency [31]. However, apples have a long generation time, with a 3 year delay from tissue culture to production of mature fruit. This time frame, the size of transgenic apple plants and the high costs in maintenance of transgenic plants precludes the production of large numbers of transgenie apple fruit for phenotypic characterisation.

Science

‘)5

122 (1997) 91-99

of an apple

ACC-oxidase

cDNA

Colour development (days from breaker (mean & S.E.. II = 9) 7.0 6.3 5.8 8.0 5.4 6 7.2 14

to 50”;1 red)

& 0.6 + 0.5 & .i + I.3 + 0.4 f 0.6 & 0.8 + 1.8

The study reported here represents an in vivo test of the effectiveness of a specific gene construct that could be used to reduce ethylene synthesis in commercial apple cultivars. The introduction of the pGAAP4 construct into tomato produced plant lines which showed reduced ethylene production amongst the primary transformants. These results indicate that the same pGAAP4 construct should also be effective in reducing ethylene production in ripening. transgenic apple fruit. This work also demonstrates that heterologousantisense RNA expressed in tomato fruit can produce a phenotypic effect. In this case, the function of the pAP4 cDNA [4] in encoding ACCoxidase had already been confirmed [32]. However, the approach of transforming tomato with antisense RNA derived from other fruits could be extended to assist in the elucidation of the function of genes with no available tomato homologue. We analysed six different tomato lines, and report varying degrees of reduction in ethylene production, with one line showing a reduction of more than 95%. This result compares fdvourably with the first homologous-antisense ACC-oxidase experiments reported, where ethylene production in the primary transformants was reduced by 87% [9]. The effects on ripening of reduced ethylene production in antisense-ACC-oxidase tomato fruit have already been studied in considerable detail [11.12]. Therefore, our work did not include extensive evaluation of postharvest quality traits. although we did observe a delay in colour development in fruit from line Al. The observation

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A

I,1 (1997) 91-99

WT

Al

Br +2 +4 +6

Br +2 +4 +6

A2 Br +2 +4 +6

A3 Br +2 +4 +6

(i) Sense RNA (ii) rRNA

(iii) Normalised signal

B

Ill

WT

Al

Br +2 +4 +6

Br +2 +4 +6

(i) Antisense RNA

A2 Br +2 +4 +6

A3 Br +2 +4 +6

I

(ii) rRNA (iii) Normalised signal Fig. 4. RNA gel blot analysis of sense tomato ACC-oxidase RNA (Panel A) and antisense pAP4 RNA (Panel B) during ripening of wild-type (WT) tomato fruit, and fruit of three independent transgenic lines (Al -3). Following strand-specific RNA hybridisation (i), each blot was stripped and hybridised with a ribosomal probe (ii), to correct for loading differences. Quantitative analysis of the RNA signal using densitometry software (SigmascanTM/Image from Jandel Scientific Software) is presented (iii), where data are shown as relative intensity, normalised to the rRNA present each lane.

that these fruit did go on to ripen suggests that the reduced level of ethylene production was still sufficient to elicit ripening responses. Apple and tomato ACC-oxidase cDNAs share 74% identity at the nucleotide level [4]. This level of homology has proved sufficient for the heterologous antisense RNA to have an effect in down-regulating the endogenous tomato ACC-oxidase RNA, in some transgenic lines. A sequence lineup of the ACC-oxidase RNAs from tomato and apple indicates there are mismatches spread throughout the duplex, and the longest region showing a perfect match is only 17 base pairs (data not shown). Thus, any RNA hybrid pro-

duced in these transgenic fruit would represent a heteroduplex, where matching regions are intermingled with short, unpaired loops. The molecular mechanism by which antisense RNA brings about a down-regulation of sense-gene expression is still being debated [33]. However, oligonucleotides of as few as 15 bases have been shown to have a down-regulatory effect (eg. [34]), and here, a longer, heterologous antisense transcript with a number of matching regions is also effective. Antisense RNA showing sequence divergence of around 20% has been shown to have an effect in some other systems e.g. antisense petunia chalcone synthase in tobacco [35].

Antisense RNA may interact with the endogenous sense message (presumably via base pairing) to promote the degradation of that message. Thus. expression of antisense RNA is usually accompanied by a decrease in level of target message. Such a decrease was observed in our transgenie line Al. but has not always been observed with heterologous antisense RNA effects. In some cases there was no associated change in the level of sense message [22,23], even though differences in enzyme activity were observed. These observations led to a suggestion that the effect of the heterologous antisense RNA may occur at the level of translation [22]. In the experiment reported here, the line with the biggest reduction in ethylene showed major reductions in the levels of steady-state RNA. This suggests that, at least in this case, the antisense effect occurs before translation. Nuclear run-off experiments are required to determine whether the effect is on the gene transcription or on stability of the sense RNA. Here. we found high steady-state levels of antisense RNA in the transgenic Al tomato fruit. This is in contrast with the findings of Hamilton et al [9] where the antisense pTOM13 primary transformants had undetectable levels of antisense RNA in ripening fruit. However, antisense pTOM13 RNA did accumulate in both the homozygous or hemizygous progeny from these same plants [33], and while co-suppression of antisense RNA has been detected in some cases, in others antisense RNA has been easily detected (eg. [10,36]). In one of our lines (A3), the antisense message was expressed at lower levels, producing only a slight reduction in sense-strand expression (Fig. 4A) and ethylene production. but in another (A2) high levels of antisense expression were not accompanied by reductions in sensestrand expression or ethylene production. This lack of correlation between amount of antisense RNA and the suppression of sense-RNA transcripts has been reported elsewhere (eg. [35]). suggesting factors such as the position of integration may be critically important [36]. In conclusion. the work presented here demonstrates that constructs of antisense genes of heterologous origin can effectively alter gene expression in ripening tomato fruit. Furthermore,

the antisense effect is not dependent on complete sequence homology, as divergence of more than 25% between the antisense gene and a tomato ‘homologue’ still produced an effect. Where fruit genes with no previously identified plant relative have been isolated, direct transformation into tomato and analysis of the phenotype should assist in the elucidation of gene function.

Acknowledgements This work Foundation ogy (CO641 part by the

was supported by a grant from The for Research. Science and Technol1). The work was also supported in ENZA International (New Zealand).

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