Colin F. Watson

Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes

Regulation of expression of specific genes by antisense RNA is a naturally occurring mechanism in bacteria1,2, although gene regulation by this mechanism has not yet been observed in higher eukaryotes. However, antisense RNA has been shown to reduce expression of specific genes when injected into frog oocytes3 and Drosophila embryos4. Inhibition of expression of artificially introduced genes has been demonstrated by transient expression of antisense RNA constructs in mammalian cells5,6, and plant protoplasts7, and by stable expression in transgenic plants8. Here, we report a striking inhibition of expression of the endogenous, developmentally regulated gene for polygalacturonase in stably transformed tomato expressing antisense RNA.

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.

Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants.

SummaryTomato plants were transformed with a chimaeric polygalacturonase (PG) gene, designed to produce a truncated PG transcript constitutively. In these plants expression of the endogenous PG gene was inhibited during ripening, resulting in a substantial reduction in PG mRNA and enzyme accumulation. This inhibition was comparable to that achieved previously using antisense genes. The expression of the truncated gene in ripe fruit was substantially lower than its expression in green fruit. Thus expression of both the endogenous and truncated genes is reduced in ripe fruit in which both are active. The implication of this observation is discussed in relation to the possible mechanism whereby sense constructs inhibit gene expression.

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants.

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.

CONTROL AND MANIPULATION OF GENE-EXPRESSION DURING TOMATO FRUIT RIPENING

Ripening is a complex developmental process involving changes in the biochemistry, physiology and gene expression of the fruit. It is an active process characterised by changes in all cellular compartments. cDNA cloning has been used as an approach to analyse changes in gene expression during fruit ripening. This has revealed that several genes are switched on specifically during fruit ripening, including one encoding polygalacturonase (PG), a major cell wall protein. These cDNA clones have been used to study the expression of the genes in normal and ripening mutant fruits, and under environmental stress conditions.The PG gene has been isolated and it has been demonstrated that 1450 bases 5′ of the coding region are sufficient for the tissue- and development-specific expression of a bacterial marker gene in transgenic tomatoes. Antisense RNA techniques have been developed to generate novel mutant tomatoes in which the biochemical function of this enzyme and its involvement in fruit softening has been tested.

Isolation and characterisation of a melon cDNA clone encoding phytoene synthase.

A cDNA clone (MEL5), encoding a protein homologous to phytoene synthase (PSY), has been isolated from a climacteric melon fruit cDNA library, using the tomato cDNA clone TOM5 [34] as a heterologous probe. MEL5 hybridised to a transcript of 1.65 kb which suggested that the 1.36 kb clone, isolated originally, was not full-length. The missing 5′ end was isolated by a reverse transcriptase-polymerase chain reaction (RT-PCR)-based method. This enabled the full sequence of the protein to be deduced and the cleavage site of the transit peptide for chromoplast import to be predicted. Northern analysis of RNA extracted from fruit samples of different ripening stages as well as from roots, leaves and flower petals was used to examine the expression pattern of the corresponding mRNA. The transcript corresponding to MEL5 is present at low quantities in unripe (green) fruit, reaches its highest levels when the fruit turns from green to orange and persists at lower levels during later ripening stages. A similar transcript was also detected in flower petals and in trace amounts in leaves and roots. Genomic Southern analysis indicates that the clone is homologous to a low-copy-number gene family. Sequence analysis showed a high degree of conservation among plant PSYs.

Isolation of a Ripening and Wound-Induced cDNA from Cucumis Melo L.With Homology to the Ethylene Forming Enzyme

Ethylene is an important plant growth regulator which is synthesized naturally during climacteric fruit ripening [Yang and Hoffmann, 1984]. The ability to extend the storage life and prevent spoilage of climacteric fruit by inhibiting ethylene biosynthesis has already been demonstrated in transgenic tomato plants expressing antisense EFE [Hamilton et al., 1990] and ACC synthase [Oehler et al., 1991] genes. In order to try and extend the storage life of melon fruit by inhibiting ethylene synthesis our initial aim was to isolate an EFE clone from a climacteric melon fruit cDNA library, using the heterologous pTOM13 (EFE) probe from tomato.

Signals for Gene Expression in Ripening Tomato Fruit

cDNA clones for a number of mRNAs expressed in ripening tomato fruit have been characterised (Grierson et al., 1986a; Maunders et al., 1987; Holdsworth et al., 1987; Ray et al., 1987, 1988). One of these (pTOM 6) has been shown by DNA and protein sequence analysis to encode the enzyme polygalacturonase (PG) (Grierson et al., 1986b), which is synthesised de novo during ripening (Tucker and Grierson, 1982) and is involved in the solubilisation of the pectin fraction of fruit cell walls. cDNA clones for PG have also been isolated by a number of other groups (DellaPenna et al., 1986; Sheehy et al., 1987; Lincoln et al., 1987). Ethylene stimulates the accumulation of ripening-related mRNAs (Maunders et al., 1987) and PG mRNA is inhibited by silver ions (Davies et al., 1988), which are thought to interfere with ethylene perception or action. Production of PG and, to a lesser extent, other mRNAs is substantially reduced in the tomato ripening mutants rin and Nr (DellaPenna et al., 1987; Knapp et al., 1989), and is also inhibited in normal fruit at 35°C (Picton and Grierson, 1988).