In order to circumvent the detrimental effects of constitutive cytokinin overexpression, Gan and Amasino (1995) devised a strategy, based on autoregulated cytokinin production, which delayed leaf senescence in transgenic tobacco without altering plant phenotype. This strategy exploited the highly senescence-specific promoter, SAG12, from a gene encoding a cysteine proteinase of A. thaliana (Lohman et al., 1994) fused to the ipt gene (synonym tmr gene) from A. tumefaciens (Hidekamp et al., 1983). The chimaeric PSAG12-IPT gene was activated only at the onset of senescence in the lower mature leaves of tobacco. This resulted in cytokinin biosynthesis in the leaves, which inhibited their senescence and, consequently, attenuated activity of the PSAG12-IPT gene, preventing overproduction of cytokinin. Whilst, theoretically, the feedback system should be tightly regulated, there are reports that the PSAG12-IPT strategy may not be so tightly autoregulated as was first expected in tobacco (Nicotiana alata) (Schroeder and Stimart, 1998) and lettuce (Lactuca sativa) (McCabe et al., 2001).
To date, the most detailed studies of the effect of ipt expression in PSAG12-IPT transgenic plants has been in a limited number of members of the Solanaceae, such as tobacco (N. tabacum) (Gan and Amasino, 1996; Schroeder and Stimart, 1998; Jordi et al., 2000), although there are reports of the introduction of Psagi2-IPT into rice (Oryza sativa) (Fu et al., 1998), cauliflower (Brassica oleracea) (Nguyen et al., 1998), A. thaliana (Zhang et al., 2000) and lettuce (Garratt et al., 2000, 2001a; McCabe et al., 2001). In particular, this strategy has been successful in the lettuce cv. Evola, delaying senescence during plant development and following harvesting of mature heads. Thus, in four homozygous transgenic lines assessed, senescent leaves were not present on any plants at the seedling stage or during later development. This trait was stably inherited over the three successive seed generations evaluated. In contrast, all corresponding azygous plants and non-transformed plants regenerated from leaf explants exhibited senescent basal leaves. Additionally, apart from retardation of leaf senescence, mature 60-day-old plants, corresponding to the age of plants from which heads are normally harvested commercially, were morphologically normal with no significant differences in head diameter or fresh weight of their leaves and roots. Following harvesting of heads at 60 days after seed sowing and storage for 7 days, the outer leaves of the heads of plants of the four homozygous PSAG12-IPT transformed plants retained their chlorophyll. In contrast, the outer leaves of heads from plants of the four azygous lines were yellow and necrotic after this storage period.
There are a number of potential applications of delayed senescence in Psag12-IPT modified lettuce. Since leaves retain their chlorophyll longer after harvesting, the most obvious application is extended post-harvest quality. Interestingly, homozygous plants also showed a significant reduction in susceptibility to infection by Botrytis cinerea (W.J.R.M. Jordi, unpublished) as this pathogen normally targets senescing tissues. Additionally, lettuce plants transformed with the PSAG12-IPT gene remained green even when nitrates became depleted in the compost. On this evidence, it was therefore proposed that the expression of this transgene might also provide a strategy for reducing the nitrate content in cultivated lettuce. In this respect, removal of nitrogen from the growth medium 5 or 10 days before harvest of PSAG12-IPT-transformed lettuce plants could result in up to 70% reduction in nitrate content with only a slight reduction in growth and no loss of leaf pigmentation and, hence, visual quality. Limits on the nitrate content of lettuce, particularly in Northern Europe, dictate that a reduced nitrate content is an important breeding objective for this crop (Gunes et al., 1994).
In addition to the SAG12 promoter, other senescence-specific promoters, such as SAG529 and SAG766A, have been used in the construction of chimaeric genes as part of a delayed senescence strategy for extending shelf-life. Such chimaeric genes have been used to transform broccoli (B. oleracea), resulting in the retardation of senescence, as measured by chlorophyll retention, following four days of post-harvest storage (Chen et al., 2001). Chimaeric ipt genes constructed using heat-shock promoters have also been used to delay senescence in an attempt to extend shelf-life (Medford et al., 1989; Smart et al., 1991; Smigocki, 1991; Ainley et al., 1993; Van Loven et al., 1993; Harding and Smigocki, 1994; Veselov et al., 1995; Cooper et al., 1995, 1996; Kudoyarova et al., 1999). However, the heat-shock process itself can affect growth and endogenous cytokinin concentrations (Van Loven et al., 1993; Wang et al., 1997a,b).
In other investigations, the 35S promoter from cauliflower mosaic virus (CaMV) has been used to control the ipt gene in transgenic tobacco and cucumber (Cucumis sativus) (Smigocki and Owens, 1988; Makarova et al., 1997a,b). However, in these cases, the constitutive expression of the ipt gene resulted in developmental abnormalities, including stunted growth and sterility.
Abnormalities have also been observed in plants transformed with the ipt gene with other constitutive promoters. For example, when a chalcone synthase promoter (PCHS) from Antirrhinum majus was used to drive the ipt gene in transgenic tobacco (Wang et al., 1997a,b), transgene expression caused inhibition of root development, retardation of leaf senescence, elevation of chlorophyll levels and a delay in flower development and, as a consequence, the onset of flowering. Expression of the PCHN-IPT gene also resulted in thicker stems resulting from concomitant enhancement of both cell division and cell expansion. In this respect, such phenotypic abnormalities are similar to those apparent during the later stages of development of tobacco and lettuce transformed with the PSAG12-IPT gene (Gan and Amasino, 1996; Jordi et al., 2000; Garratt et al., 2000, 2001a; McCabe et al.,
2001), and are consistent with the overproduction of endogenous cytokinins. Cell enlargement observed in PSAG12-IPT and PCHN-IPT plants could be due to increases in water uptake, resulting from increased osmotic pressure. Such an increase in osmotic pressure would be consistent with sugar (hexose) accumulation characteristic of PSAG12-IPT plants (Garratt et al., 2000, 2001a; McCabe et al., 2001). Tobacco transformed with a copper-inducible ipt gene (Cu-IPT) exhibited delayed senescence when treated with physiological concentrations of Cu2+ (McKenzie et al., 1998).
Delayed leaf senescence has also been achieved in transgenic tobacco, using the homeobox gene, knotted1 (kn1), isolated from A. thaliana, fused to the senescence-specific promoter, pSAG12 (Ori et al., 1999). Normally, the kn1 gene and its homologues are expressed in shoot meristems. Interestingly, the PSAG12-kn1 transformed plants exhibited delayed senescence with no significant developmental abnormalities. In addition to the delayed senescence phenotype, there were a number of other characteristics of these plants, which were also observed in PSAG12-IPT transformed tobacco plants, the most striking of which was a significant increase in cytokinin concentrations in the leaves. It was proposed that kn1 may act as a transcription factor, mediating the accumulation of cytokinin (Ori et al., 1999). Similarly, in the lettuce cv. Luxor, expression of PetE-KNAT1, an Arabidopsis kn1 -like homologue under the control of the pea plastocyanin promoter PetE, also resulted in a delay in leaf senescence (Frugis et al., 2001).
During the onset of leaf senescence and fruit ripening, plasma membranes as well as the membranes of the endoplasmic reticulum, lose their selective permeability and fluidity (Hong et al., 2000), such changes being known to initiate programmed cell death (Thompson et al., 2000). This loss of selective permeability has been attributed to molecular perturbations in the lipid bilayers, resulting from the increase in the ratio of non-esterified to esterified fatty acids in the membranes. The de-esterification of these fatty acids is caused by the action of senescence-induced lipase (lipolytic acyl hydrolase) (Thompson et al., 2000). Furthermore, de-esterification of polyunsaturated fatty acids acts as a substrate for the action of lipoxygenase, which results in lipid peroxidation and, hence, progressive membrane rigidity and loss of functional integrity (Asada and Takahashi, 1987).
Transgenic plants of A. thaliana have been generated in which the expression of senescence-induced lipase has been down-regulated through the constitutive expression of the full length gene in its antisense orientation, under the regulation of a 35S promoter (Thompson et al., 2000). The resulting plants exhibited delayed leaf senescence, demonstrating that manipulation of lipase expression could also be an effective strategy for extending shelf-life.
In flowers, the antisense inhibition of the 1-aminocyclopropane oxidase gene in the carnation cvs. Red Sim and White Sim delayed petal senescence in trans-genic plants (Savin et al., 1994), potentially extending vase-life. This inhibition of senescence corresponded to a significant reduction in endogenous 1-aminocyclopropane oxidase and ACC synthase mRNAs. Similarly, antisense inhibition of the 1-aminocyclopropane oxidase gene in tomato has been demonstrated to delay the onset and rate of fruit ripening (John et al., 1995; Bolitho et al., 1997). More recently, a rab11/YPT3 homologue from tomato, encoding a gua-nacine tryphosphate (GTPase), believed to be involved in the control of protein trafficking within cells, has been down-regulated in transgenic tomato, using antisense inhibition (Lu et al., 2001). Fruit from plants expressing the antisense gene had normal pigmentation, but failed to develop a soft texture.
The manipulation of antioxidant biosynthesis in lettuce has been achieved using a construct consisting of chimeric genes encoding elements of the ascor-bate-glutathione pathway (Garratt et al., 2001b). Overexpression of these transgenes enhanced the oxyradical scavenging potential and antioxidant content of transgenic plants. Homozygous plants exhibited up to a six-fold increase in foliar reduced glutathione compared to their azygous controls. Foliar hydrogen peroxide was up to three-fold lower in the upper leaves and up to two-fold lower in the middle and lower leaves of homozygous plants, compared to controls. Lipid peroxidation was also significantly decreased, indicating that membrane integrity was maintained. Furthermore, leaf discs excised from transgenic plants and floated on water for 7 days to induce senescence, expressed foliar hydrogen peroxide concentrations which were 40% lower than those concentrations detected in leaf discs excised from azygous (control) plants. The chlorophyll content of intact 60-day-old transgenic plants was significantly (P < 0.05) higher in the upper and lower leaves (>40% and 20%, respectively). As well as improving crop performance during growth, the stimulation of antioxidant capacity, which delayed peroxidation, enhanced the post-harvest performance of the transgenic lettuce plants, with an extension of shelf-life, together with an improvement in appearance and nutritional content.
In addition to delaying visible signs of senescence, attempts have been made to reduce or to delay the generation of off-flavours associated with the storage of food products. This is being achieved by the inactivation or inhibition of the enzymes responsible for producing such undesirable products, or by developing transgenic plants deficient in the undesirable enzyme(s). For example, improvement in the flavour, stability and hence shelf-life of preparations of soybean (Glycine max), specifically soy flour and soy milk, has been achieved by the removal of the enzyme lipoxygenase-2 (LOX-2) (Davies et al., 1987).
In the case of tomato (Lycopersicon esculentum) and tobacco, expression of the yeast D-9 desaturase transgene increased the concentration of most mono-unsaturated fatty acids in both these plants. Additionally, this decreased the concentration of saturated fatty acids in tomato (Polashock et al., 1992; Wang et al., 1996), leading to changes in the flavour profile of fruits of the transgenic plants. However, whilst this demonstrates the ability to alter flavour profiles by the genetic manipulation of fatty acids, this approach has not, as yet, been applied directly to extending shelf-life. In contrast, significant increases have been observed in the shelf-life of fruits of transgenic plants of tomato with antisense suppressed polygalacturonase activity (Sozzi-Quiroga and Fraschina, 1997). As well as being less susceptible to damage and infection, the transgenic tomato fruits exhibited retarded over-ripening, but maintained normal development during pre-senescence. Sensory, physicochemical and biochemical monitoring indicated that standard preference ratings, as used by retail outlets, for these transgenic fruits were significantly superior compared to those of non-transformed plants, particularly in terms of fruit colour and flavour (Sozzi-Quiroga and Fraschina, 1997).
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