Regulation of ripening and senescence

Tomato is a climacteric fruit and ripening is naturally regulated by ethylene produced by the fruit at the onset of ripening. Two transgenic approaches have been used to reduce endogenous ethylene production in ripening fruit in order to delay the onset and rate of fruit ripening. The pathway of ethylene biosynthesis is now well known (Kende, 1993; Barry et al., 2000) and the final two steps in the pathway, conversion of S-adenosyl methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) and its oxidization to ethylene have been targeted for modification in transgenic plants. One approach has been to metabolize either SAM or ACC to an inactive product and the second approach has been to specifically suppress the expression of the two ethylene biosynthetic enzymes required to catalyze the final steps in the pathway. Both approaches resulted in fruit with significantly reduced ethylene content and greatly delayed ripening.

To metabolize the ethylene precursors, SAM and ACC, microbial genes have been expressed in tomato. Metabolic inactivation of SAM was carried out by transgenic expression of a bacteriophage T3 S-adenosylmethionine hydrolase (SAMase) gene in tomato (Good et al., 1994). The SAMase enzyme converts Sadenosyl methionine, to methylthioadenosine and homoserine rather than ACC, and serves as a means to divert SAM from the ethylene biosynthetic pathway. The transgenic tomato plants were engineered to express SAMase only in ripening fruit after the breaker stage by linking the T3 SAMase coding sequence to the E8 promoter (Deikman et al., 1992), a tomato fruit-specific and ripening regulated promoter. Consequently, the effects of reduced ethylene were observed only in ripening fruit and these fruit exhibited delayed ripening and enhanced firmness (Good et al., 1994). An alternative strategy was to express a microbial gene encoding ACC deaminase, an enzyme that converts ACC to α-ketoglutarate in transgenic tomato.

The ACC deaminase enzyme was identified in a strain of Pseudomonas that utilized ACC as a nitrogen source, and the corresponding gene was introduced into tomato as a means to divert ACC from the ethylene biosynthetic pathway. The transgenic fruit exhibited reduced ethylene production and the fruit ripened at a slower rate and remained firm (Klee et al., 1991). No effects were observed in the vegetative tissues of these transgenic plants and, surprisingly, fruit ripening was delayed only in fruit detached from the plant but not in fruit allowed to ripen on the plant (Klee, 1993). Interestingly, transgenic tomato lines expressing a bacterial ACC deaminase with the root-specific RolB promoter or the pathogen inducible tobacco promoter P(RB-1b) were able to grow in the presence of heavy metals such as Cd, Co, Cu, Ni, Pb and Zn (Grichko et al., 2000).

Transgenic tomato plants with reduced ethylene levels also were developed by suppressed expression of endogenous genes encoding the ultimate and penultimate steps in the biosynthetic pathway. Constitutive expression of a tomato fruit-specific ACC synthase gene in its sense orientation resulted in two phenotypically different groups of plants, those over-expressing the ACC synthase gene, and those in which ACC synthase gene expression was reduced by co-suppression of the endogenous gene (Lee et al., 1997). The transgenic lines with reduced ACC synthase gene expression exhibited reduced ethylene production and reduced ripening.

Another transgenic approach to reduce ethylene in ripening fruit relied on expression of antisense genes to suppress the expression of the endogenous ACC oxidase or ACC synthase genes. This approach was first explored as a means to deduce the function of a tomato gene (pTOM13) by antisense suppression of its expression (Griersonet al., 1990). Fruit from these plants produced considerably reduced amounts of ethylene and ripened more slowly. Subsequently, pTOM13 was shown to encode the ethylene-forming enzyme of tomato, ACC oxidase (Hamilton et al., 1991; Spanu et al., 1991). This initial finding was later expanded to demonstrate that constitutive expression of the tomato antisense ACC oxidase gene caused delayed fruit ripening (Piconet al., 1995) and delayed leaf senescence by 10 to 14 days (Johnet al., 1995). A cDNA encoding apple fruit ACC oxidase has also been expressed in its antisense orientation in tomato, resulting in greater than 95% reduction in the endogenous tomato ACC oxidase mRNA accumulation, a reduction in the ethylene production, and delayed ripening (Bolitho et al., 1997). Theologis and colleagues also demonstrated that constitutive expression of an antisense ACC synthase gene significantly reduced endogenous ethylene production and delayed fruit ripening (Oeller et al., 1991). Using these transgenic plants, the requirement for ethylene to initiate and maintain the progression of ripening and senescence was demonstrated.

Altering the plants’ perception of ethylene is another approach that has been taken to modify the role of ethylene in transgenic tomato plants. The primary ethylene receptor is encoded by the family of ETR genes, which in tomato consists of five members Le(ETR1, LeETR2, Nr, LeETR4 and LeETR5) that collectively control ethylene sensitivity throughout the plant. These genes encode histidine kinase sensors homologous to two-component signaling proteins found in bacteria and in Arabidopsis. LeETR4, Nr and LeETR5 are expressed in ripening fruit and their expression is stimulated by ethylene, suggesting that the ETR genes are important in fruit for both the competence to respond to ethylene and the specific tissue responses to ethylene. Expression of a mutant form of Nr in tomato renders the plants insensitive to ethylene (Wilkinson et al., 1995). Fruit ripening was also delayed in transgenic tomato plants with antisense suppressed expression of Nr(Tieman et al., 2000), although in this case suppression of Nr is compensated, at least in part, by an increase in LeEXP4 expression. Paradoxically, suppression of LeETR4 expression in transgenic tomato results in increased ethylene sensitivity and premature flower senescence and more rapid fruit ripening. Ethylene regulation of  ETR gene expression is complex, but future transgenic lines of tomato with specifically designed expression or suppression of multiple members of ETR the family may provide the basis to produce plants and tissues that exhibit precise responses to both endogenous and exogenous ethylene.