The parent organism is Carica papaya L. (papaya, or paw paw), which is exotic to Australia, but is grown both commercially and in domestic gardens in tropical and subtropical parts of Australia from Western Australia to New South Wales. More detailed information on papaya can be found in a review document ‘The Biology and Ecology of Papaya (paw paw), Carica papaya L., in Australia’ that was produced in order to inform this risk assessment process. This document is available at the OGTR website (http://www.ogtr.gov.au).
Section 3 The introduced genes
Section 3.1 The capacs 1 and capacs 2 genes
The capacs 1 and capacs 2 genes occur naturally in non-GM papayas (Mason & Botella 1997). They encode the enzyme ACC synthase, a component of the ethylene biosynthesis pathway (see Section 1.1). Ethylene is a plant hormone that regulates many aspects of plant growth and development, including fruit ripening (Alexander & Grierson 2002). ACC synthase (1-amino-cyclopropane-1-carboxylic acid synthase) catalyses the synthesis of ACC (1-amino-cyclopropane-1-carboxylic acid), a metabolic intermediate required for the production of ethylene. The capacs 1 and capacs 2 genes introduced into the GM papayas proposed for release have been isolated from the Carica papaya variety ‘Solo’ and are re-introduced into the same variety in different forms (see Table 1) to down-regulate papaya’s production of ethylene during fruit development.
Down-regulating ACC synthase using the technique employed by the applicant has previously been shown to prevent fruit ripening in tomato (Oeller et al. 1991). The inhibition of ripening is reversed if external ethylene is applied to the tomato fruit. Similarly, the down-regulation of ACC oxidase, another enzyme necessary for the production of ethylene, has been shown to prevent ripening in rock melon (cantaloupe) (Ayub et al. 1996). The aim of the proposed release is to examine the effect of down-regulating capacs 1 and capacs 2 on the ripening of papaya fruit under normal field conditions.
The capacs 1 gene is active primarily at the initial stages of fruit ripening, while the capacs 2 gene is most highly active later in ripening (Mason & Botella 1997). The expression pattern of capacs 2 in ripening papaya fruit is similar to that of a tomato ACC synthase gene known to be crucial to ripening of tomato fruit (Oeller et al. 1991; Mason & Botella 1997). However, the expression pattern of capacs 1 is unusual for a ACC synthase gene. This is because, unlike capacs 2 and other common ACC synthase genes such as leacs 2 from tomato fruit (Lincoln et al. 1993), capacs 1 is expressed at high levels in mature green papaya fruit, but its expression decreases steadily during ripening (Mason & Botella 1997).
The ACC synthase genes targeted in the GM papaya plants are only expressed in fruit, during the ripening process (information provided by the applicant). Thus, the applicant has indicated that down-regulating capacs 1 and capacs 2 is not expected to affect any other ethylene-related processes in the plant. However, as the introduced ACC synthase genes are under the control of the 35S promoter (see Section 3.5), it is possible that ethylene production may also be down-regulated in plant tissues other than fruit, such as leaves or seed. For example, in melons genetically modified to down-regulate ethylene biosynthesis via modification of the activity of another enzyme involved in ethylene biosynthesis, ACC oxidase, ethylene production is also down-regulated in leaves (Ayub et al. 1996).
Future applications to release these GM papayas (which would be subject to separate applications and assessments) would require information regarding the impact of down-regulating ethylene biosynthesis on key ethylene-related processes in papaya other than fruit ripening, such as plant growth and development or disease susceptibility, before the application could be considered.
3.1.1 Mechanisms for altering capacs 1 and capacs 2 activity
Two different methods of initiating down regulation of gene activity are used to generate GM papaya plants with altered ACC synthase activity. Together, these methods are known as ‘gene silencing’, as they result in ‘silencing’ of the normal activity of a targeted gene present in the plant.
With the first method, silencing of the papaya’s capacs 1 and capacs 2 genes is achieved by inserting the genes into the papaya in either the sense or antisense orientation. This means that the inserted genes will be either in the correct (sense) orientation for translation of the gene to produce the protein, or in the opposite (antisense; incorrect) orientation. The presence of genes inserted in the sense or antisense orientation can silence both the inserted gene and the copy of the gene already present in the plant. This phenomenon was first identified in plants in 1990 (Napoli et al. 1990; Van der Krol et al. 1990) and has since been extensively used in the analysis of plant genes (Wang & Waterhouse 2002).
Gene silencing via the introduction of sense or antisense copies of several different genes involved in ethylene synthesis and other aspects of fruit ripening has been demonstrated in other fruit such as tomato and melon (for example (Oeller et al. 1991; Smith et al. 1990; Ayub et al. 1996). In Australia, the limited and controlled release of pineapples with silencing of ACC synthase via insertion of a truncated version of the pineapple ACC synthase gene in a sense orientation was authorised by the previous voluntary system under deemed licence PR-95 and an application to continue evaluation of this release has been lodged with the Regulator.
The second method of achieving silencing of the papaya ACC synthase activity involves a more recent technique that introduces both a sense and an antisense copy of a native gene into a plant, linked together by a short non-coding DNA sequence. Such genetic constructs are known as ‘hairpins’. Genes that are introduced in this way are more effective at silencing genes than introducing either the sense or antisense gene alone (Waterhouse et al. 1998). The capacs 1 and capacs 2 genes are also introduced into the papaya in this way, using a piece of non-coding DNA from the pdk (pyruvate orthophosphate dikinase) gene from the plant Flavaria trinervia, to link the sense and antisense versions of the genes. Silencing of another gene involved in ethylene synthesis, ACC oxidase (1-aminocyclopropane-1-carboxylate oxidase), by a similar technique has been demonstrated in tomato (Hamilton et al. 1998).
Regardless of which method initiates gene silencing, the ultimate result is decrease in expression of the target gene. Gene silencing in plants appears to be caused by mechanisms that exist naturally to control gene expression and to defend against plant viruses. Recently, many details of the functioning of these mechanisms have been revealed (Waterhouse et al. 2001; Vaucheret et al. 2001).
Section 3.2 The etr1-1 gene
The etr1 gene encodes for a receptor protein that is involved in ethylene perception (Chang et al. 1993). etr1 was identified in the plant, Arabidopsis thaliana, by analysing plants that are insensitive to ethylene. These plants have a non-functional version of the etr1 gene (etr1-1) and lack several normal responses to ethylene, such as promotion of seed germination, inhibition of root and hypocotyl elongation and acceleration of leaf senescence (Bleecker et al. 1988). The ETR1 protein in these plants lacks a functional ethylene binding site and is, thereby, unable to perceive the presence of ethylene (Schaller & Bleecker 1995).
The non-functional etr1-1 gene is a dominant gene. This means that plants carrying one copy of the functional gene (etr1) and one copy of the non-functional gene (etr1-1) are insensitive to ethylene (i.e. the effect of the non-functional gene over-rides that of the functional gene).
The non-functional Arabidopsis etr1-1 gene confers ethylene insensitivity on plants other than Arabidopsis when it is introduced (Wilkinson et al. 1997). This effect has been demonstrated for tomato, petunia (Wilkinson et al. 1997), tobacco (Knoester et al. 1998) and carnations (Bovy et al. 1999). Plants carrying the non-functional etr1-1 gene exhibit delayed floral senescence and delayed fruit ripening (Wilkinson et al. 1997; Knoester et al. 1998; Bovy et al. 1999).
Plants carrying the etr1-1 gene do not show major differences in overall growth and development, prior to flowering, compared to plants carrying a functional copy of the gene (Bleecker et al. 1988; Knoester et al. 1998; Bovy et al. 1999). However, some characteristics other than fruit ripening and floral senescence may be altered in GM ethylene insensitive plants. Arabidopsis plants with a copy of the etr1 1 gene are similar to normal Arabidopsis plants, except they have lower seed germination rates, altered growth patterns in germinating seeds, slightly larger and longer-lived leaves and lower peroxidase activity (Bleecker et al. 1988). These plants also produce more ethylene under certain conditions, due to lack of feedback inhibition of the ethylene production pathway. Tobacco plants carrying the Arabidopsis etr1-1 gene also have altered growth morphology of germinating seeds, a lower rate of leaf senescence and impaired perception of neighbouring plants and produce higher levels of ethylene (Knoester et al. 1998).
3.2.1 Ethylene perception and plant disease resistance
In addition to its role in plant growth and development, ethylene is also involved in plant responses to stresses, such as wounding or pathogen attack (Stepanova & Ecker 2000; Thomma et al. 2001). The role of ethylene in plant resistance to pathogens appears to be complex and is not fully understood. Ethylene acts as a signal in initiating plant disease resistance responses and is also associated with symptom development (Stepanova & Ecker 2000). Ethylene insensitive plants have been observed to display both enhanced susceptibility and enhanced resistance to different pathogens.
The ability of plants carrying the non-functional etr1-1 gene, and other genes conferring ethylene insensitivity, to mount resistance responses to pathogens has been investigated. Arabidopsis plants with loss of ability to perceive ethylene due to loss of function in the ein2 gene may be more susceptible to disease caused by a variety of fungal and bacterial pathogens (Thomma et al. 1999; Norman-Setterblad et al. 2000; Ton et al. 2002) and are unable to mount an induced systemic resistance response normally triggered by contact with non-pathogenic soil bacteria (Knoester et al. 1999). Similarly, tobacco plants carrying the Arabidopsis etr1-1 gene may exhibit decreased levels of some defence-related proteins, and suffer disease caused by soil-borne fungi that normally have limited ability to infect tobacco plants (Knoester et al. 1998; Geraats et al. 2002).
In contrast to these observations, plants with impaired ethylene perception may also suffer reduced disease symptoms when attacked by normally virulent pathogens. For example, symptom development is reduced in some ethylene-insensitive Arabidopsis plants infected with pathogenic bacteria (Bent et al. 1992). Tomato plants with impaired ethylene perception or ethylene synthesis also show decreased symptoms in response to some pathogenic bacteria and a fungus that are normally able to cause disease (Lund et al. 1998). Likewise, ethylene-insensitive Arabidopsis plants have been observed to be less susceptible to a nematode that is normally able to parasitise these plants (Wubben et al. 2001).
GM papaya plants with altered ethylene synthesis or ethylene perception could potentially have altered responses to both pathogenic and non-pathogenic microorganisms. Such altered responses could manifest either in non-fruit parts of plants with constitutive down-regulation of ethylene production or potentially in the fruit in etr1-1 expressing plants. It is expected that etr1-1 will only be expressed in GM papaya fruit tissues as the promoter controlling its expression is fruit-specific (see Section 3.5 of this Appendix). Observations made on other ethylene-insensitive plants suggest that the effect of altered ethylene metabolism on the resistance response of papaya to microorganisms encountered in the environment may not be predictable and is best elucidated by field evaluation.
The possibility that altering the response of GM papayas to pathogens may affect the weediness of GM papayas is considered in Appendix 3.
Section 3.3 The uidA gene
The uidA gene, from the common soil bacterium Escherichia coli, codes for the enzyme -glucuronidase (GUS). The GUS enzyme converts a colourless substrate into a blue colour in a simple laboratory assay and is used as a reporter or ‘marker’ to detect tissues that have been successfully genetically modified. Exposure of plant tissues containing the GUS gene to this substrate facilitates measurement of the expression of the uidA gene (Jefferson et al. 1986).
If expression of the uidA gene is controlled by the promoter from another gene, the strength and distribution of the blue colour in the plant tissue indicates the strength of the promoter and hence expression levels of the other gene can be inferred. In the proposed release, GUS is used to evaluate the efficiency of the transformation vector containing the CaMV 35S promoter (see Section 3.5 for details) for driving expression of the capacs 1 and capacs 2 genes in papaya tissues.
The nptII gene was isolated from the bacterial Tn5 transposon (Beck et al. 1982). It encodes the enzyme neomycin phosphotransferase type II (NPTII) which confers resistance to aminoglycoside antibiotics such as kanamycin and neomycin. The NPTII enzyme uses ATP to phosphorylate neomycin, and the related kanamycin, thereby inactivating the antibiotic and preventing it from killing the NPTII producing cell.
The nptII gene functions as a selectable marker in the initial laboratory stages of papaya plant cell selection following genetic modification, allowing modified cells to grow while inhibiting the growth of non-GM cells.
The process of the biolistic method of transformation may result in the introduction of additional genetic elements or genes from the transformation vector to the GM papaya plants. Some of the genetic elements or genes present in the vector are designed for replication of the vector in bacteria in the laboratory and will not have any function in plants. Other genes in the vector are designed for selection of bacterial cells carrying the vector in the laboratory. The GM papayas proposed for release are likely to contain either an additional copy of the kanamycin resistance gene (nptII) or a copy of a gene encoding resistance to the antibiotic ampicillan (bla). These genes are under the control of bacterial promoters and will not be expressed in the GM papaya plants. Some plants will also carry a portion of the -galactosidase (Lac Z) gene, which produces an enzyme used for selection of E. coli carrying the vector in the laboratory. The Lac Z gene will not be functional in the GM papaya plants.
Future applications (which would be subject to separate applications and assessments) to release these GM papayas would require information regarding the presence in the GM papayas of other genes from the vector, before the application could be considered.
The potential toxicity of the introduced ETR1-1, antibiotic resistance and GUS proteins and ACC synthase enzymes and the potential of these genes to transfer to other organisms, are discussed in Appendices 2 and 4, respectively.
Section 3.5 Regulatory sequences
Expression of the capacs 1, capacs 2, and uidA genes is under the control of a promoter (a region of DNA that determines whether a gene is expressed and to what extent) from the cauliflower mosaic virus 35S gene (the 35S promoter). It is likely that use of this promoter in the GM papayas will result in expression of these genes in all plant tissues. A mRNA termination region, including a polyadenylation signal, is also required for gene expression in plants, and is provided by the nos terminator (from the Agrobacterium nopaline synthase gene).
Expression of the etr1-1 gene is under the control of a promoter from an apple (Malus domestica) polygalacturonase (pga) gene (Atkinson et al. 1998). In GM tomatoes, this promoter has been used to target expression of introduced genes in ripening fruit (Atkinson et al. 1998). Use of this promoter in the GM papayas proposed for release by UQ is expected to result in the etr1-1 gene being expressed only in ripening fruit and not in other parts of the GM papaya plant.
Future applications to release these GM papayas would require information regarding the tissues in which genes under the control of the pga promoter are expressed.
The nptII gene for selection in the laboratory is under the control of the nos-promoter and the nos-terminator (both derived from the Agrobacterium nopaline synthase gene).
Although some of the regulatory sequences transferred to the GM papaya plants are derived from plant pathogens, they only represent a very small proportion of the pathogen genome. The sequences are not, in themselves, infectious or pathogenic.