As discussed in Chapter 8, the effects of ethylene in fruit-ripening and flower senescence are of particular interest in agriculture. In climacteric fruits such as apple, pear, tomato, banana, papaya, mango, and avocado, an increase in respiration stimulated by ethylene accompanies the start of the ripening process. It is thought that at the molecular level ethylene coordinates the expression of the genes that participate in ripening (Gray et al.,
1992). The protein products of these genes are presumably involved in causing the changes that take place during ripening such as changes in fruit color (due to chlorophyll degradation and carotenoid pigment synthesis), development of sweetness (due to breakdown of starch into sugars or sugar translocation from other parts of the plant) and softening of the fruit (due to changes in cell wall components).
The understanding of basic aspects of ethylene biology has lagged behind the manipulation of ethylene as a horticultural practice. However, the pathway for ethylene biosynthesis is known (Fig. 6-2; also see Chapter 8) and more recently advances have been made in elucidating how ethylene signal transduction occurs. The signal transduction pathway initiates with the receptor which was identified in 1995 (Schaller and Bleecker, 1995). There are also several ethylene signaling mutants available and these are providing new insights into how the ethylene signal transduction pathway works beyond the receptor. Currently ethylene is the best understood plant hormone system at the molecular level. These recent advances in basic aspects of ethylene biology have also triggered several attempts to manipulate senescence using genetic engineering approaches.
Most attempts to block ethylene-mediated senescence have involved manipulating ethy-lene biosynthesis or perception. This has been accomplished by reducing ethylene synthesis by "turning off" ethylene-synthesizing enzymes or by reducing ethylene levels by depletion of ethylene precursors. More recently, a dominant negative approach using a mutant ethylene receptor has been employed.
In 1991, Oeller et al. reported a successful manipulation of tomato fruit ripening by turning off ACC synthase expression. ACC synthase is encoded by a gene family, two members of which are expressed during fruit ripening, LE-ACC2 and LE-ACC4. The cDNA for LE-ACC2 was placed in reverse orientation in front of the strong constitutive CaMV 35S promoter and transformed into plants. Tomato fruits from an ACC synthase antisense line that showed the strongest phenotype had ethylene production inhibited by 99.5%, and RNA blot analyses with gene-specific probes showed that mRNAs for both LE-ACC2 and LE-ACC4 were undetectable in the transgenic fruits. The red color characteristic of fruit ripening did not develop and these fruits never softened or produced a ripe aroma. Treatment with exogenous ethylene was able to reverse this phenotype leading to fruits that were indistinguishable from wild-type ripe fruits.
The turning off strategy has also been tried with the last enzyme in the pathway of ethylene synthesis, ACC oxidase (ACO). Transgenic tomato plants showing a reduction in ethylene synthesis were obtained by antisense expression of pTOM13, believed to encode a tomato ACO gene, driven by the CaMV 35S promoter (Hamilton et al., 1990). In plants expressing antisense pTOM13 mRNA, wound-induced expression of this gene was greatly reduced and its expression was undetectable in ripening fruits. In detached fruits from certain plants containing two copies of the antisense transgene, ethylene evolution was inhibited by 97%. Although the time at which fruit ripening initiated was not changed, the transgenic fruits were less susceptible to overripening and shriveling when kept at room temperature compared to control fruits. Interestingly, Picton et al. (1993) reported that the time at which the fruit is detached from the vine is related to the extent that ripening is affected. The most extreme delay in fruit ripening was observed when transgenic fruits were collected at the mature-green stage (i.e. full-sized fruit with its basal part starting to show chlorophyll loss). In addition to the fruit-related phenotype, a delay in the onset of foliar senescence was observed in the transgenic plants. At 8 weeks post-germination, control plants had leaves with advanced senescence while the transgenic counterparts had no visible signs of chlorophyll loss. Wild-type lower leaves showed approximately 40% lower chlorophyll levels than the corresponding leaves of transgenic tomato plants. This delay in leaf senescence is similar to results obtained with the ethylene-insensitive Arabidopsis mutant etr1-1. Etr1-1 leaves live 30% longer than wild-type indicating that ethylene plays a role in the timing of leaf senescence but that ultimately senescence occurs in the absence of ethylene perception (Grbic and Bleecker, 1995).
An antisense version of an ACO gene from melon has been introduced into Charentais cantaloupe which has poor storage capability (Ayub et al., 1996). In one transgenic line a 99% reduction in ethylene production was observed in fruits. Interestingly, the transgenic fruits did not abscise from the plant because there was no activation of the peduncular abscission zone. At the time of commercial harvest, transgenic fruits were more than twofold firmer than wild type. Exogenous ethylene application was able to restore yellowing of the rind, softening of flesh, and abscission zone activation in the peduncule.
Studies in Dianthus caryophyllus (carnation) illustrate the effects of ethylene in flower senescence (Nichols et al., 1983). After pollination, the petals of carnation flowers wilt in 1 to 2 days. Petal senescence is accompanied by an increase in respiratory activity and a climacteric-like increase in ethylene production. By 3 hours after pollination there is a 10-fold increase in ethylene production by stigmas. Petals increase ethylene production later. Application of ethylene inhibitors is able to retard petal senescence. Senescing carnation petals exhibit a characteristic "inrolling" behavior which also occurs in response to exogenous ethylene. This inrolling can be delayed by inhibitors of ethylene synthesis. The gene for ACO from petunia was cloned and used to generate transgenic carnation plants that express an antisense ACO gene under the control of a strong constitutive promoter (Savin et al., 1995). The transgenic carnations did not display the typical inrolling behavior. While the normal vase life of the cultivars used is approximately 5 days from harvest to inrolling, some of the transgenic flowers had their vase life increased up to 9 days.
Ethylene levels can also be reduced by the expression of enzymes that metabolize ethylene precursors. A Pseudomonas strain that is able to use ACC as its sole nitrogen source for growth was identified (Klee etal, 1991). From this strain, the gene responsible for the ACC-degrading activity was cloned and found to encode ACC-deaminase, which degrades ACC to ammonia and alpha-ketobutyric acid. The ACC-deaminase gene was then introduced into tomato plants under the control of the CaMV 35S promoter. Transgenic plants expressing ACC-deaminase were phenotypically indistinguishable from wild-type plants. However, their fruits showed a significant delay in the progression of ripening and reduced ethylene synthesis (Klee, 1993).
Another study utilized the S-adenosylmethionine hydrolase (SAMase) from bacterio-phage T3 (Good et al., 1994). SAMase degrades SAM to methylthioadenosine and homoserine, lowering levels of the metabolic precursor to ethylene. This approach has been successful in tomato plants where SAMase expression was achieved in a tissue- and developmental stage-specific manner directed by the E8 promoter. This regulated expression of SAMase was necessary to avoid disturbing other pathways in other tissues such as DNA methylation, polyamine and phospholipid biosynthesis that involve SAM. The E8 promoter has been shown to be ethylene inducible and specifically activated during tomato fruit ripening (Lincoln et al., 1987). Transgenic tomato fruits at the time of picking had ethylene levels similar to controls. However, their ability to produce ethylene declined at later timepoints such that ethylene synthesis was reduced by approximately 80% and the fruits took twice as long as controls to reach the final ripened stage.
An innovative way to manipulate senescence involving a dominant negative approach arose from the finding that the ETR gene encodes an ethylene receptor in Arabidopsis (Schaller and Bleecker, 1995). A mutated version of ETR, encoded by the etr1-1 allele, confers dominant ethylene insensitivity in Arabidopsis. When etr1-1 is heterologously expressed in petunia and tomato plants there is a significant delay of flower senescence and abscission and fruit ripening (Wilkinson et al., 1997). For example, the corolla of a transgenic petunia line remained turgid for at least 8 days while wild-type flowers collapsed by the third day. Interestingly, it was found that in transgenic petunia plants the ethylene production that normally follows the pollination of petunia flowers exceeded that of wildtype. This suggests that there is a feedback mechanism to control ethylene production after pollination involving the wild-type ethylene receptors, and that this feedback was disrupted by the transgene expression. Similar to that which was observed with transgenic tomato fruits obtained by the approaches described above, detached tomato fruits from transgenic lines expressing etr1-1 remained yellow even after 3 months whereas the wild-type control fruits turned red, softened and eventually rotted.
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