Abstract
The next wave of genetically engineered crops will use genes that modify gene regulation, plant metabolism, or signal transduction. The potential for these genes to have cascading effects on metabolism, physiology, and development increases the possibility for unintended effects that influence crop performance or environmental impact. This review examines altered ethylene signaling as an example of a complex trait with many horticultural applications. Genes for modified ethylene production or perception intended to regulate ripening, senescence, or stress or disease resistance have been observed to cause a broad range of secondary effects, including modified growth and development and increased severity to biotic and abiotic stresses. Successful use of complex traits in crop varieties will frequently require methods to reduce secondary effects, including the use of targeted gene expression. Risk assessment will need to consider observed pleiotropic effects on fitness within the context of potential environmental impacts.
The commercialization and widespread cultivation of genetically engineered crops has grown rapidly in the 10 years since their initial introduction. More than 114 million ha were planted worldwide in 2007 (James, 2007). The vast majority of these crops (greater than 99%) are engineered for two traits, herbicide resistance and/or insect resistance using Bacillus thuringiensis (Bt) cry genes. In these crops, the engineered genes encode protein products that directly confer the trait of interest. Herbicide resistance genes either encode an herbicide-insensitive target molecule or an enzyme that degrades the herbicide (Sahora et al., 1998). Insect resistance is conferred by Bt cry genes that encode proteins toxic to certain species of insects (Carpenter et al., 2002). Despite expression throughout the plant, the Bt and herbicide resistance genes are not involved in modifying or regulating plant growth, development, or response to the environment and therefore do not generally cause pleiotropic (i.e., secondary) effects. Phenotypic changes other than direct effects of these traits have been primarily associated with the site of gene insertion, and those affecting performance are selected against by plant breeders in the early stages of crop development (Mihaliak, 2002).
A much broader range of traits and variety of genes are under currently under development by public researchers and private companies throughout the world. These include genes engineered to confer increased stress resistance, enhanced disease resistance, modified metabolism, and altered growth and development (Nickson, 2008; Wolfenbarger and Grumet, 2002; USDA-APHIS records). Such modifications could allow for cultivation of crops under adverse or marginal environmental conditions, reduce chemical inputs, or tailor plants for nutritional or industrial needs. Unlike genes used in the first wave of transgenic crops, these types of genes are intended to modify gene regulation or metabolic or signaling pathways of the plant. Multiple steps can occur between the protein product (e.g., transcription factor) and the ultimate desired phenotype (e.g., stress resistance). As a result, the activities of many additional gene products can be affected, increasing the possibility for pleiotropic effects that could cause ecologically relevant changes in phenotype (Wolfenbarger and Grumet, 2002).
In this review, we use modified ethylene production, perception, or response, collectively referred to as ethylene signaling, as an example of an engineered trait that can result in multiple effects, both intended and unintended. There is an extensive body of literature describing a diverse range of roles for ethylene throughout plant growth and development, and ethylene-related genes have been used to introduce a variety of traits in numerous species (Table 1). Plant processes influenced by ethylene include seedling development, root and shoot growth, flower development, senescence, fruit ripening, and responses to abiotic and biotic stresses (Abeles et al., 1992). Thus, altering ethylene signaling for a characteristic such as increased flower longevity may, in turn, result in secondary changes in abiotic stress responses or disease resistance. We discuss approaches used to modify ethylene production or perception, intended phenotypic effects, and examine the complexity of pleiotropic effects with respect to potential ecological impact.
Examples of genetically modified ethylene signaling for agronomic or horticultural performance.
Modification of Ethylene Signaling
Modification of the production or response to ethylene to manipulate plant growth and development is not a new concept. Ethylene-releasing compounds and inhibitors of ethylene synthesis and perception have been broadly used in agriculture to control multiple facets of crop production. The ethylene-releasing compound, ethephon (2-chloroethylphosphonic acid), has been used to retard growth and prevent lodging in wheat and barley, initiate uniform flowering in pineapple, induce female sex expression in cucurbits, increase latex flow in rubber, increase bud hardiness and bloom delay in sweet cherry and plum, reduce curing time in tobacco, promote ripening in banana and tomato, and promote fruit abscission in cotton and defoliation in tree species (Abeles et al., 1992; Arshad and Frankenberger, 2002; Hedden and Phillips, 2000; Yang and Oetiker, 1998). Ethylene production can be inhibited by treatment with aminoethyloxvinylglycine or aminooxyacetic acid; ethylene response is inhibited by silver-containing compounds, 2,5-norbornidiene, or 1-methylcyclopropene (Abeles et al., 1992; Sisler and Serek, 1997). Applications of these compounds include delay of fruit ripening, prevention of flower senescence or fruit abscission, and increased shelf life of leafy vegetables (Arshad and Frankenberger, 2002). Given the range of applications, it is not surprising that modified ethylene production or response has been a subject of numerous genetic engineering efforts (Czarny et al., 2006; Stearns and Glick, 2003).
The ability to engineer altered ethylene synthesis or signaling requires knowledge of underlying molecular mechanisms and cloning of key ethylene-related genes. Ethylene is synthesized from the widely used methyl donor, S-adenosyl methionine (S-AdoMet), in two steps (Yang and Hoffman, 1984). The first committed step in the pathway is conversion of S-AdoMet to 1-aminocyclopropane carboxylate (ACC) by the enzyme ACC synthase (ACS); in the second step, ACC is oxidized to ethylene by ACC oxidase (ACO). The ACS and ACO genes have been cloned from multiple species and are generally encoded by multigene families whose members are differentially regulated by a variety of developmental, hormonal, or environmental signals (Bleecker and Kende, 2000).
The path of ethylene perception and signaling also is well characterized (Wang et al., 2002; Fig. 1). A family of receptors (ETR1, ETR2, ERS1, ERS2, and EIN4) homologous to two component histidine kinases are responsible for binding ethylene (Bleecker and Kende, 2000; Johnson and Ecker, 1998). Mutations that eliminate ethylene binding result in dominant, ethylene-insensitive phenotypes. In the absence of ethylene, the receptors activate CTR1 (constitutive ethylene response), a Ser/Thr protein kinase, which negatively regulates downstream ethylene responses (and when mutated results in constitutive ethylene response). In the presence of ethylene, CTR1 is deactivated, allowing the positive regulator EIN2 (ethylene-insensitive) to promote expression of members of the EIN3 transcription factor family. EIN3 induces expression of the ethylene-responsive element binding factor ERF1 (and other members of the ERF or ethylene response element binding protein family), which, in turn, regulate expression of genes responsible for various ethylene responses (Bleecker and Kende, 2000; Wang et al., 2002). The ERF family includes both transcriptional activators (e.g., ERF1) and repressors (ERF3, ERF4), which when overexpressed, confer constitutive ethylene response or insensitivity, respectively (Fujimoto et al., 2000; Yang et al., 2005).
Schematic representation of key components of the ethylene signaling pathway. Asterisks indicate genes targeted for modification of ethylene perception or response by genetic engineering.
Citation: HortScience horts 44, 1; 10.21273/HORTSCI.44.1.94
Approaches to modulate ethylene production or activity have included up- or downregulation of ethylene biosynthetic genes ACS and ACO; introduction of genes encoding enzymes to reduce ethylene production (e.g., ACC deaminase, S-AdoMet hydrolase); introduction of mutant ethylene receptor genes (e.g., etr1-1); and introduction of the ERF family transcription factor genes (Table 1).
Four plants engineered for modified ethylene signaling have received regulatory approval for environmental release, which is a necessary prerequisite to commercial production. These include: a carnation engineered for increased cut flower life through sense suppression of ACS in Australia and the European Union (http://www.agbios.com); and tomato lines engineered for delayed ripening either by a truncated ACS gene or by introduction of SAM hydrolase or ACC deaminase (ACD) in the United States (http://www.agbios.com). A wider range of crops engineered with ethylene-related genes have been tested in field trials, including apple, broccoli, coffee, melon, papaya, pineapple, potato, plum, and strawberry (USDA-APHIS records). In almost all cases, the plants were engineered for decreased ethylene production to facilitate long-distance transport and extended product shelf life.
Phenotypes Associated with Modified Ethylene Signaling
Plant growth and development.
Genes for altered ethylene production or perception have been introduced to modify numerous growth and development-related traits, including root growth, nodule formation, floral development and sex expression, fruit development, ripening and quality characters, senescence, and abscission (Table 1). Based on the demonstrated effectiveness of external applications of ethylene-releasing or inhibitory compounds to regulate postharvest performance, the traits receiving the most interest for genetic engineering have been modified ripening and senescence. Fruit ripening is characterized by biochemical and physical changes in color, texture, flavor, aroma, and nutritional content (Adams-Phillips et al., 2004; Giovannoni, 2004; Gray et al., 1994). For many fruits, termed climacteric such as tomato, banana, apple, and peach and pear, ripening is initiated by a burst in ethylene production. The burst in ethylene is associated with numerous changes in gene expression, including changes in signal transduction and transcription factors as well as genes associated with processes such as softening, aroma, and pigment production (e.g., Alba et al., 2005; Schaffer et al., 2007).
Reduced expression of the ethylene biosynthetic enzymes ACS and ACO, or the introduction of ACD or SAM hydrolase, has been used to retard ripening in tomato and melon (Good et al., 1994; Guis et al., 1997; Klee, 1993; Reed et al., 1995; Silva et al., 2004; Sozzi et al., 2001) and modify aroma and improve fruit quality in apple (Dandekar et al., 2004; DeFilippi et al., 2004, 2005; Schaffer et al., 2007). Changes observed in the transgenic fruits include reduced ester, alcohol, and volatile production; higher titratable acidity; reduced malic acid degradation; modified sugar content; increased soluble solids; increased firmness; reduced cell wall degradation; and reduced chlorophyll degradation (Dandekar et al., 2004; DeFilippi et al., 2004, 2005; Flores et al., 2001; Guis et al., 1997; Schaffer et al., 2007; Silva et al., 2004; Sozzi et al., 2001).
Senescence and abscission involve complex biochemical processes whereby catabolism, mobilization of metabolites, and physical disintegration lead to programmed cell death (Chandlee, 2001). Mutant ethylene receptor or regulator etr1, ERS, and EIN2 genes have been used to delay senescence in petunia, chrysanthemum, Nemesia strumosa, and Campanula carpatica flowers (Aida et al., 1998; Bovy et al., 1999; Clevenger et al., 2004; Cui et al., 2004; Narumi et al., 2005; Shaw et al., 2002; Shibuya et al., 2004; Sriskandarajah et al., 2007; Wilkinson et al., 1997) and delay abscission in tomato fruit (Whitelaw et al., 2002). ACD, antisense ACO, and mutant ers genes have been used to retain postharvest quality (reduce chlorophyll loss) in broccoli (Chen et al., 2004; Gapper et al., 2005; Henzi et al., 2000). The inhibited ethylene signaling was associated with downregulation of cysteine protease, metallothionein-like protein, hexokinase, invertase, and sucrose transporters (Gapper et al., 2005). These examples indicate how modification of a single gene can cause a host of changes in gene expression, metabolism, and physiology necessary to produce the intended phenotype.
Interactions of ethylene with other plant hormones has been extensively demonstrated, leading to a complex network of overlapping signals and responses (Gazzarrini and McCourt, 2003; Rashotte et al., 2005; Swarup et al., 2002; Visser and Voesenek, 2005). One of the most frequently observed examples of interaction involves auxin and ethylene, which coordinately regulate several processes, including apical hook formation, root growth, and hypocotyl phototropism (Swarup et al., 2002). Individual members of ACS gene families exhibit increased expression in response to auxin and, in several cases, possess upstream auxin response element sequences (Johnson and Ecker, 1998; Swarup et al., 2002; Woeste et al., 1999). Auxin can also influence stability of the ACS and ACO enzymes (Chae et al., 2000; Chae and Keiber, 2005).
Other hormones that interact with ethylene include gibberellins with respect to seed dormancy and growth responses (Brady and McCourt, 2003) and brassionosteroids, which can cause increased ethylene production and enhance ethylene-associated responses such as ripening, apical hook formation, and female sex expression in cucurbits (Arteca et al., 1988; Papadopoulou and Grumet, 2005; Sasse, 2003). In contrast, jasmonic acid and abscisic acid can antagonize ethylene signaling (Beaudoin et al., 2000; Ellis and Turner, 2001; Vardhini and Rao, 2002; Woeste et al., 1999). In many cases, interactions with other hormones were uncovered when genetic screens intended to identify genes associated with one hormone identified genes in other hormone pathways (Brady and McCourt, 2003). These observations demonstrate the interrelatedness among hormone effects and potential for modified ethylene production or perception to influence a broad range of responses.
Response to abiotic and biotic stresses.
Ethylene also plays important roles in plant responses to abiotic and biotic stresses. Increased ethylene production has been observed in response to temperature extremes, salinity, shade, drought, flooding, ozone, and heavy metal contamination (Morgan and Drew, 1997). In some species, stress-induced ethylene production is associated with increased growth or modified development to allow for stress avoidance. Elevated ethylene can cause more acute leaf angles to optimize light capture under shade conditions or petiole or internode elongation in response to submergence (Cox et al., 2004; Kende et al., 1998; Pierek et al., 2003, 2004; Voesenek et al., 2003). Ethylene-related root responses include development of aerenchyma to facilitate oxygen transport in response in response to flooding and development of adventitious roots (Bragina et al., 2001, 2003; McDonald and Visser, 2003; Visser and Voesenek, 2005). Overexpression of the ethylene and the osmotic stress-inducible responsive factor from tomato, TERF1, which binds to both ethylene and osmotic stress response elements, increases salt and drought tolerance in tobacco (Huang et al., 2004; Zhang et al., 2005). Similarly, the ethylene, osmotic, and jasmonic acid response factor from tomato confers increased salt tolerance to tobacco (Wang et al., 2004).
In contrast to adaptive ethylene-mediated responses, in flood-sensitive species such as tomato, flood-induced ethylene production is associated with deleterious consequences, including epinasty, chlorosis, and necrosis (Grichko and Glick, 2001). Introduction of a root-specific ACD gene to reduce ethylene production caused increased flood tolerance in tomato as measured by increased growth, leaf chlorophyll content, and reduced epinasty (Grichko and Glick, 2001). Similarly, decreased tomato leaf and shoot growth caused by compacted soil was reversed by reduced ethylene production through expression of antisense ACO (Hussain et al., 1999). Constitutive or root-specific expression of ACD in canola conferred increased growth in nickel-contaminated soil (Stearns et al., 2005). Transgenic tobacco expressing ACD acquired higher levels of metals (cadmium, cobalt, copper, nickel, lead, zinc) in their tissues but was less subject to the deleterious effects than its nontransgenic counterparts (Grichko et al., 2000). Transgenic tobacco, potato, tomato, or birch with suppressed expression of an ozone-inducible ACS gene or an introduced ACD or mutant etr1-1 gene was more ozone-tolerant (Nakajima et al., 2002; Sinn et al., 2004; Vahala et al., 2003).
These studies demonstrate that the effects of ethylene on plant stress responses vary depending on the species and environment. In each case, ethylene production was induced in response to the stress. However, the nature of the downstream events, and whether they cause beneficial or injurious effects, appears to be dependent on the range of adaptive responses of individual species and thus the introduction of ethylene-related transgenes can have differential stress-related responses depending on the species.
Ethylene also plays important roles in plant defense, including induction of the hypersensitive response, pathogenesis-related (PR) genes, and other defense genes (Okubara and Paulitz, 2005; Rojo et al., 2003). Increased ethylene signaling can confer increased disease resistance and decreased ethylene can cause decreased resistance. Overexpression of the TERF1-positive regulator in tobacco and tomato led to constitutive PR gene expression and enhanced resistance to Ralstonia solanacearum (Zhang et al., 2004). Increased sensitivity to ethylene in antisense LeETR4 tomato plants resulted in stronger and more rapid PR gene expression and more rapid hypersensitive response after inoculation with avirulent populations of Xanthomonas campestris (Ciardi et al., 2001). Loss of ethylene sensitivity in tobacco conferred by mutant ethylene receptor etr1 caused loss of nonhost resistance against normally nonpathogenic fungi and decreased resistance to several soilborne or necrotropic pathogens (Geraats et al., 2003; Knoester et al., 1998).
The results of modified ethylene signaling can be variable depending on the host–pathogen combination. Reduced ethylene sensitivity has been associated with reduced severity of bacterial spot symptoms for transgenic tomatoes expressing ACD despite equivalent pathogen levels, presumably as a result of reduction of ethylene-related necrosis responses (Lund et al., 1998). Similarly, transformation of tomato with ACD led to reduced ethylene production and reduced symptom severity in response to Verticillium infection despite the presence of the pathogen (Robison et al., 2001). Ethylene insensitivity caused increased resistance to Peronospora tabacina in tobacco but did not affect the hypersensitive response to Tobacco mosaic virus (Geraats et al., 2003; Knoester et al., 1998). This range of responses, including both increased and decreased resistance, indicates the complexity of predicting the effect of modulating ethylene responses on specific diseases and further indicates that the same gene in the same species may cause different responses, depending on the pathogen.
As was noted for plant development, ethylene does not act alone. Plant defense responses involve a complex network of signaling involving salicylic acid (SA), jasmonic acid (JA), and ethylene. Deciphering this network is the subject of current research in many laboratories and several recent reviews (e.g., Bostock, 2005; Glazebrook, 2005; Jalali et al., 2006; Pozo et al., 2004; Rojo et al., 2003). JA and ethylene frequently act synergistically to confer tolerance to necotrophic root pathogens such as Pythium, Rhizoctonia, and Phytophthora. It was suggested that the effects of JA and ethylene on root development may lead to altered balance between root growth and defense gene expression (Glazebrook, 2005; Okubara and Paulitz, 2005). The outcome of interaction with SA signaling has been contradictory. Depending on the pathogen, expression of the tomato ERF in Arabidopsis was either positively regulated by JA and ethylene or by SA, indicating antagonism between the pathways. It has been suggested that responses to the different hormones can be antagonistic, cooperative, or synergistic to balance the metabolic costs of defense with the ability to deter opportunistic agents that may be able to invade should initial infection become successful. (Bostock, 2005; Rojo et al., 2003). Transcriptional elements for JA, SA, and ethylene often reside with the same defense gene promoter, presumably allowing fine-tuned responses to various pathogens (Jalali et al., 2006)
Pleiotropic Effects of Modified Ethylene Signaling
Given the wide variety of ethylene-mediated developmental processes, disease and stress responses, and interaction with other hormone responses, it is not surprising that alteration of ethylene for one purpose results in secondary effects on other phenotypes, especially if the introduced genes are expressed at all times throughout the plant as is the case with genes driven by a constitutive promoter. Studies cited in Table 1 show numerous cases in which multiple effects were observed in addition to the specific intended trait. Accompanying changes included modified volatile production, phenolic regulation, effects on ABA and polyamine levels, modified fruit set patterns, fruit ripening and abscission, modified branch and root structure, reduced plant and flower size, increased sensitivity to abiotic stresses, and increased susceptibility to insects and diseases (Table 1).
For example, transgenic ethylene-insensitive tomato, petunia, and melon plants engineered for flowering and ripening traits exhibited reduced root mass and reduced ability to be propagated by cuttings (Clark et al., 1999; Clevenger et al., 2004; Gubrium et al., 2000; Klee, 2002; Little et al., 2007; Shibuya et al., 2004). The ethylene-insensitive plants also exhibited difficulty in penetrating heavy soils and increased sensitivity to water stress, possibly as a result of reduced root mass and/or generally increased sensitivity to abiotic stresses (Clark et al., 1999; Little et al., 2007; Shibuya et al., 2004). In at least two cases, when subjected to stressful environments, decreased ethylene production or perception was associated with plant death. Transgenic etr1-1 and EIN2 petunias exhibited premature plant death that was more frequent at stress points such as transfer to soil when exposed to high temperature conditions or when grown in the field (Shibuya et al., 2004). A large portion of the petunia plants carrying a mutant ERS gene did not survive; the loss was attributed to increased disease susceptibility (Shaw et al., 2002).
Secondary effects were also observed for traits influencing reproductive development. Expression of a mutated ethylene receptor gene in tobacco led to an alteration in floral structure; the ethylene-insensitive lines exhibited heterostyly with the stigma located above the anthers (Takada et al., 2005). Because heterostyly is viewed as a mechanism to promote outcrossing, changes of this sort could influence expected levels of gene flow. Indeed, the heterostylus transgenic tobacco lines exhibited greatly reduced numbers of seeds unless they were artificially pollinated (Takada et al., 2005). In the most severely affected lines, however, even artificially pollinated fruit produced less seed, suggesting effects on both mechanism of pollination and fertility. Pollen grains in the most severe lines failed to develop properly. For petunias, the effects of ethylene insensitivity were primarily observed on reduced seed size and viability rather than reduced seed set and pollen viability (Clevenger et al., 2004). It was suggested that the effects on seed size, which were maternally controlled, resulted from altered ripening processes that might influence seed development processes. The seed viability was directly correlated to seed size.
Modified ethylene signaling also had unexpected effects on carpel-bearing flower development in transgenic melons (Papadopoulou et al., 2005). The observed increase in initiation of carpel-bearing buds on ACS-overexpressing lines was predicted based on prior studies demonstrating the promotive effect of exogenous ethylene on female sex expression. Analysis of flower development, however, indicated that ethylene has a second role and is necessary for completed maturation of the carpel-bearing bud to anthesis (Papadopoulou et al., 2005). Transgenic melons expressing mutant etr1 under a carpel-directed promoter further demonstrated the requirement of ethylene for maturation of carpel-bearing flowers to anthesis (Little et al., 2007). Fruit set patterns also were affected. Typically, melons will not set fruit at closely spaced nodes. However, ethylene overproducing lines exhibited a fivefold increase in closely spaced fruits (Grumet et al., 2007; Papadopoulou et al., 2005), suggesting that increased ethylene modified internal signaling normally involved in regulating resource allocation.
These results also show that the observed secondary effects can vary, depending on the physiology of the species, and also provide valuable new insights into plant developmental processes. Reduced expression of the LeETR1 transcript in tomato affected fruit abscission and internode length, but not ripening characteristics, suggesting specificity among ethylene receptors with respect to different ethylene-mediated processes (Whitelaw et al., 2002). Inhibited ethylene perception caused different phenotypes in species in the same family, heterostyly and reduced pollen viability in tobacco, and reduced seed size and viability in petunia (Clevenger et al., 2004; Takada et al., 2005).
Further assessment of the complexity of effects and interactions can be obtained from global gene expression studies. Gene expression profiles of ethylene mutants, and control and ethylene-treated wild-type Arabidopsis plants, showed that 3% to 7% of ≈6000 tested genes were regulated by ethylene (DePaepe et al., 2004; Van Zhong and Burns, 2003). Among the differentially expressed genes were ethylene biosynthetic and signaling components, transcription factors, components of other hormone pathways, primary metabolic proteins, disease and defense-related proteins, and many of unknown function. Overlaps were observed among responses to ethylene, ABA, auxin, and sugar (DePaepe et al., 2004). Analysis of the affected genes may provide new insight into possible secondary effects that may arise from altered ethylene signaling.
Implications for Environmental Risk Assessment
It is clear from the previously cited examples that introduction of genes for modified ethylene signaling can cause a broad range of effects, including both intended phenotypes and unintended secondary effects. From a risk assessment standpoint, key questions are: what is the ecological relevance of these changes and are there ways to minimize such changes? Possible ecological impacts would result if the engineered crop, or interfertile wild relatives who become recipients of the engineered gene, exhibit changes causing them to become weedy in agricultural fields, invasive in natural environments, or otherwise disrupt the surrounding ecosystem (Conner et al., 2003; Dale et al., 2002; Ellstrand, 2001; Snow et al., 2005). These environmental concerns need to be addressed whether we are dealing with a complex engineered trait, a simple engineered trait such as Bt-mediated insect resistance, or a conventionally bred trait (e.g., disease resistance). The aspect that is different with a more complex engineered trait such as altered ethylene signaling, relative to a simple engineered trait, is the range of phenotypes potentially affected. Although examples of secondary effects have been occasionally reported for the current commercialized transgenes [outcrossing rates in herbicide resistant Arabidopsis (Bergelson et al., 1998); increased lignin in Bt maize (Saxena and Stotzky, 2001)], these effects are much more limited than the broad range and frequency of occurrence of modified phenotypes summarized in Table 1.
Pleiotropic effects associated with complex traits could be potentially advantageous or detrimental. For a trait such as ethylene signaling, which modulates a multitude of developmental and environmental responses, constitutive modification often results in maladapted plants [e.g., loss of disease resistance or water stress tolerance (Clark et al., 1999; Shaw et al., 2002; Shibuya et al., 2004)] resulting from disruption of finely tuned, interdependent processes. Such plants will not be of use for commercial production and so are not of concern with respect to ecological risk assessment consideration.
Successful engineering of complex traits in crop varieties will frequently require methods to minimize secondary effects. One method to reduce secondary effects is to use specific promoters that mediate tissue-specific, developmentally specific, and/or environmentally responsive gene expression. Fruit- or root-targeted gene expression has been tested for some ethylene-related traits, including modified ripening and resistance to flooding or heavy metals (Good et al., 1994; Grichko and Glick, 2001; Stearns et al., 2005). Use of restricted promoters will also reduce the risk of potential ecological impacts of the transgenic crop.
Of critical importance in assessing the potential ecological impact of a GE crop is whether the introduced gene and its associated phenotype confers a fitness advantage, a prerequisite for increased weediness or invasiveness (Conner et al., 2003; Dale et al., 2002; Hancock, 2003). Direct comparisons of performance are routinely made in greenhouse and confined field trials between the engineered line and its equivalent nontransgenic counterpart of an array of factors related to fitness such as plant vigor, flowering time, reproductive capacity, and susceptibility to pathogens and insects (Mihaliak, 2002). Potential for gene exchange with compatible relatives should also be evaluated; where crosscompatible wild populations coexist with the crop, the fitness impact of the transgene should be assessed in the genetic background of the relative (Burke and Riesberg, 2003; Campbell et al., 2006; Snow et al., 2003). If the transgene can confer a fitness advantage, especially in native settings that may not be observed under cultivation (e.g., nutrient limited or routinely water-logged soils), studies should be performed in natural environments or conditions that simulate those environments. Nickson (2008) emphasizes the importance of focusing on situations in which there could be an adverse effect on native communities.
In summary, ethylene plays critical roles in coordinating plant growth and development and response to biotic and abiotic stresses. These roles make ethylene signaling an attractive target for genetic engineering, but also can result in numerous pleiotropic effects. Targeted gene expression can potentially minimize secondary effects that influence successful crop performance or ecological impact. Risk assessment will need to consider the full range of pleiotropic changes within the context of potential ecological impacts.
Literature Cited
Abeles, F.B., Morgan, P.W. & Saltveit M.E. Jr 1992 Ethylene in plant biology 2nd Ed Academic Press San Diego, CA
Adams-Phillips, L., Barry, C. & Giovannoni, J. 2004 Signal transduction systems regulating fruit ripening Trends Plant Sci. 9 331 338
Aida, R., Yoshida, T., Ichimura, K., Goto, R. & Shibata, M. 1998 Extension of flower longevity in transgenic torenia plants incorporating ACC oxidase transgene Plant Sci. 138 91 101
Alba, R., Payton, P., Fei, Z., McQuinn, R., Debbie, P., Martin, G.B., Tansksley, S.D. & Giovannoni, J.J. 2005 Transcriptome and selected metabolite analyses reveal multiple points of ethylene regulatory control during tomato fruit development Plant Cell 17 2954 2965
Arshad, M. & Frankenberger, W.T. 2002 Ethylene agricultural sources and applications Kluwer Academic/Plenum Publishers New York, NY
Arteca, R.N., Bachman, J.M. & Mandava, N.B. 1988 Effects of indole-3-acetic acid and brassinosteroid on ethylene biosynthesis in etiolated mung bean hypocotyl segments J. Plant Physiol. 133 430 435
Ayub, R., Guis, M., Ben-Amor, M., Gillot, L., Roustan, J.P., Latché, A., Bouzayen, M. & Pech, J.C. 1996 Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits Nat. Biotechnol. 14 826 866
Beaudoin, N., Serizet, C., Gosti, F. & Graudat, J. 2000 Interaction between abscisic acid and ethylene signaling cascades Plant Cell 12 1103 1115
Ben-Amor, M., Flores, B., Latche, A., Bouzayen, F., Pech, J.C. & Romojaro, F. 1999 Inhibition of ethylene biosynthesis by antisense ACC oxidase RNA prevents chilling injury in Charentais cantaloupe melons Plant Cell Environ. 22 1579 1586
Bergelson, J., Purrington, C.B. & Wichmann, G. 1998 Promiscuity in transgenic plants Nature 395 25
Bleecker, A.B. & Kende, H. 2000 Ethylene: A gaseous signal molecule in plants Annu. Rev. Cell Dev. Biol. 16 1 18
Bostock, R.M. 2005 Signal crosstalk and induced resistance: Straddling the line between cost and benefit Annu. Rev. Phytopathol. 43 545 580
Bovy, A.G., Angenent, G.C., Dons, H.J.M. & van Altrost, A. 1999 Heterologous expression of the Arabidopsis etr1-1 allele inhibits the senescence of carnation flowers Mol. Breed. 5 301 308
Brady, S.M. & McCourt, P. 2003 Hormone cross-talk in seed dormancy J. Plant Growth Regulat. 22 25 31
Bragina, T.V., Martinovich, L.I., Rodionova, N.A., Bezborodov, A.M. & Grineva, G.M. 2001 Ethylene-induced activation of xylanase in adventitious roots of maize as a response to the stress effect of root submersion Appl. Biochem. Microbiol. 37 618 621
Bragina, T.V., Rodionova, N.A. & Grineva, G.M. 2003 Ethylene production and activation of hydrolytic enzymes during acclimation of maize seedlings to partial flooding Russ. J. Plant Physiol. 50 794 798
Burke, J.M. & Riesberg, L.H. 2003 Fitness effects of transgenic disease resistance in sunflower Science 300 1250
Campbell, L.G., Snow, A.A. & Ridley, C.E. 2006 Weed evolution after crop gene introgression: Greater survival and fecundity of hybrids in a new environment Ecol. Lett. 9 1198 1209
Carpenter, J., Feliot, A., Goode, T., Hammig, M., Oristad, D. & Sankula, S. 2002 Comparative environmental impacts of biotechnology-derived and traditional soybean, corn, and cotton crops Council Agr. Sci. Technol Ames, IA
Chae, H.S., Cho, Y.G., Park, M.Y., Lee, M.C., Eun, M.Y., Kang, B.G. & Kim, W.T. 2000 Hormonal cross-talk between auxin and ethylene differentially regulates the expression of two members of the 1-aminocyclopropane-1-carboxylate oxidase family in rice (Oryza sativa L.) Plant Cell Physiol. 41 354 362
Chae, H.S. & Keiber, J.J. 2005 Eto brute? Role of ACS turnover in regulating ethylene biosynthesis Trends Plant Sci. 10 291 296
Chandlee, J.M. 2001 Current molecular understanding of the genetically programmed process of leaf senescence Physiol. Plant. 113 1 8
Chen, L.F.O., Huang, J.Y., Wang, Y.H., Chen, Y.T. & Shaw, J.F. 2004 Ethylene insensitive and post-harvest yellowing retardation in mutant ethylene response sensor (boers) gene transformed broccoli (Brassica oleracea var. italica) Mol. Breed. 14 199 213
Ciardi, J.A., Tieman, D.M., Jones, J.B. & Klee, H.J. 2001 Reduced expression of the tomato ethylene receptor gene LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv. vesicatoria Mol. Plant Microbe Interact. 14 487 495
Clark, D.G., Gubrium, E.K., Barrett, J.E., Nell, T.A. & Klee, H.J. 1999 Root formation in ethylene-insensitive plants Plant Physiol. 121 53 59
Clevenger, D.J., Barrett, J.E., Klee, H.J. & Clark, D.G. 2004 Factors affecting seed production in transgenic ethylene-insensitive petunias J. Amer. Soc. Hort. Sci. 129 401 406
Conner, A.J., Glare, T.R. & Nap, J. 2003 The release of genetically modified crops into the environment, Part II. Overview of ecological risk assessment Plant J. 33 19 46
Cox, M.C.H., Benschop, J.J., Vreeburg, R.A.M., Wagemaker, C.A.M., Moritz, T., Peeters, A.J.M. & Voesenek, A.C.J. 2004 The roles of ethylene, auxin, abscisic acid, and gibberellin in the hyponastic growth of submerged Rumex palustris petioles Plant Physiol. 136 2948 2960
Cui, M.L., Takada, K., Ma, B. & Ezura, H. 2004 Overexpression of a mutated melon ethylene receptor gene Cm-ETR1/H69A, confers reduced ethylene sensitivity in a heterologous plant, Nemesia strumosa Plant Sci. 167 253 258
Czarny, J.C., Grichko, V.P. & Glick, B.R. 2006 Genetic modulation of ethylene biosynthesis and signaling in plants Biotechnol. Adv. 24 410 419
Dale, P.J., Clarke, B. & Fontes, E.M.G. 2002 Potential for the environmental impact of transgenic crops Nat. Biotechnol. 20 567 574
Dandekar, A.M., Teo, G., Defilippi, B.G., Uratsu, S.I., Passey, A.J., Kader, A.A., Stow, J.R., Colgan, R.J. & James, D.J. 2004 Effect of down-regulation of ethylene biosynthesis on fruit flavor complex in apple fruit Transgenic Res. 13 373 384
DeFilippi, B.G., Dandekar, A.M. & Kader, A.A. 2004 Impact of suppression of ethylene action or biosynthesis on flavor metabolites in apple (Malus domestica Borkh) fruits J. Agr. Food Chem. 52 5694 5701
DeFilippi, B.G., Kader, A.A. & Dandekar, A.M. 2005 Apple aroma: Alcohol acyltransferase, a rate limiting step for ester biosynthesis, is regulated by ethylene Plant Sci. 168 1199 1210
DePaepe, A., Vuylsteke, M., Van Hummelen, P., Zabeau, M. & Van Der Straeten, D. 2004 Transcriptional profiling by cDNA-AFLP and microarray analysis reveals novel insights into the early response to ethylene in Arabidopsis Plant J. 39 537 559
Ellis, C. & Turner, J.G. 2001 The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens Plant Cell 13 1025 1033
Ellstrand, N.C. 2001 When transgenes wander, should we worry? Plant Physiol. 125 1543 1545
Flores, F.B., Martinez-Madrid, M.C., Sanchez-Hidalgo, F.J. & Romojaro, F. 2001 Differential rind and pulp ripening of transgenic antisense ACC oxidase melon Plant Physiol. Biochem. 39 37 43
Fujimoto, S.Y., Ohta, M., Usui, A., Shinshi, H. & Ohme-Takagi, M. 2000 Arabidopsis ethylene responsive element binding factors act as transcriptional activators or repressors of GCC-mediated gene expression Plant Cell 12 393 404
Gapper, N.E., Coupe, S.A., McKenzie, M.J., Scott, R.W., Christey, M.C., Lill, R.E., McManus, M.T. & Jameson, P.E. 2005 Senescence associated down-regulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase delays harvest-induced senescence in broccoli Funct. Plant Biol. 32 891 901
Gazzarrini, S. & McCourt, P. 2003 Cross-talk in plant hormone signaling: What Arabidopsis mutants are telling us Ann. Bot. (Lond.) 91 605 612
Geraats, B.P.J., Bakker, P.A.H.M., Lawrence, C.B., Achuo, E.A., Hofte, M. & van Loon, L.C. 2003 Ethylene insensitive tobacco shows differentially altered susceptibility to different pathogens Phytopathology 93 813 821
Giovannoni, J.J. 2004 Genetic regulation of fruit development and ripening Plant Cell 16 S170 S180
Glazebrook, J. 2005 Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu. Rev. Phytopathol. 43 205 227
Good, X., Kellogg, J.A., Wagoner, W., Langhoff, D., Matsumara, W. & Bestwick, R.K. 1994 Reduced ethylene synthesis by transgenic tomatoes expressing S-adenosylmethione hydrolase Plant Mol. Biol. 26 781 790
Gray, J.E., Picton, S., Giovannoni, J.J. & Grierson, D. 1994 The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening Plant Cell Environ. 17 557 571
Grichko, V.P., Filby, B. & Glick, B.R. 2000 Increased ability of transgenic plants expressing the bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb and Zn J. Biotechnol. 81 45 53
Grichko, V.P. & Glick, B.R. 2001 Flooding tolerance of transgenic tomato plants expressing the bacterial enzyme ACC deaminase controlled by the 35S, rolD or PRB-1b promoter Plant Physiol. Biochem. 39 19 25
Grumet, R., Katzir, N.L., Little, H.A., Portnoy, V. & Burger, Y. 2007 New insights into reproductive development in melon (Cucumis melo L.) Intl. J. Plant Dev. Biol. 1 253 264
Gubrium, E.K., Clevenger, D.J., Clark, D.G., Barrett, J.E. & Nell, T.A. 2000 Reproduction and horticultural performance of transgenic ethylene insensitive petunias J. Amer. Soc. Hort. Sci. 125 277 281
Guis, M., Botondi, R., BenAmor, M., Ayub, R., Bouzayen, M., Pech, J.C. & Latche, A. 1997 Ripening-associated biochemical traits of Cantaloupe Charantais melons expressing an antisense ACC oxidase transgene J. Amer. Soc. Hort. Sci. 122 748 751
Haines, M.M., Shiel, P.J., Fellman, J.K. & Berger, P.H. 2003 Abnormalities in growth, development and physiological responses to biotic and abiotic stress in potato (Solanum tuberosum) transformed with Arabidopsis ETR1 J. Agr. Sci. 141 333 347
Hancock, J.F. 2003 A framework for assessing the risk of transgenic crops Bioscience 53 512 519
Hedden, P. & Phillips, A.L. 2000 Manipulation of hormone biosynthetic genes in transgenic plants Curr. Opin. Biotechnol. 11 130 137
Henzi, M.X., Christey, M.C. & McNeil, D.L. 2000 Morphological characterization and agronomic evaluation of transgenic broccoli (Brassica oleracea L. var. italica) containing an antisense ACC oxidase gene Euphytica 113 9 18
Huang, Z.J., Zhang, Z.J., Zhang, K.L., Zhang, H.B., Huang, D.F. & Huang, R.F. 2004 Tomato TERF1 modulates ethylene response and enhances osmotic stress tolerance by activating expression of downstream genes FEBS Lett. 573 110 116
Hussain, A., Black, C.R., Taylor, I.B. & Roberts, J.A. 1999 Soil compaction. A role for ethylene in regulating leaf expansion and shoot growth in tomato? Plant Physiol. 121 1227 1237
Jalali, B.L., Bhargava, S. & Kamble, A. 2006 Signal transduction and transcriptional regulation of plant defense responses J. Phytopathol. 154 65 74
James, C. 2007 Global status of commercialized biotech/GM Crops: 2006, ISAAA Brief No. 35 ISAAA
Johnson, P.R. & Ecker, J.R. 1998 The ethylene gas signal transduction pathway: A molecular respective Annu. Rev. Genet. 32 227 254
Kende, H., van der Knaap, E. & Cho, H.T. 1998 Deepwater rice: A model plant to study stem elongation Plant Physiol. 118 1105 1110
Klee, H.J. 1993 Ripening physiology of fruit from transgenic tomato (Lycopersicon esculentum) plants with reduced ethylene synthesis Plant Physiol. 102 911 916
Klee, H.J. 2002 Engineered changes in ethylene signal transduction pathways 81 84 Wolfenbarger L.L. Proceedings of a Workshop on Criteria for Field Testing of Plants with Engineered Regulatory, Metabolic, and Signaling Pathways 3–4 June 2002 Information Systems for Biotechnology Blacksburg, VA
Knoester, M., van Loon, L.C., van den Heuvel, J., Hennig, J. & Bol, J.F. 1998 Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi Proc. Natl. Acad. Sci. USA 95 1933 1937
Kumar, A., Taylor, M.A., Arif, S.A.M. & Davies, H.V. 1996 Potato plants expressing sense and antisense S-adenosylmethionine decarboxylase (SAMDC) transgenes show altered levels of polyamines and ethylene: Antisense plans display abnormal phenotypes Plant J. 9 147 158
Lee, J.H., Hong, J.P., Oh, S.K., Lee, S., Choi, D. & Kim, W.T. 2004 The ethylene responsive factor like protein 1 (CaERFLP1) of hot pepper (Capsicum annum L.) interacts in vitro with both GCC and DRE/CRT sequences with different binding affinities: Possible biological roles of CaERFLP1 in response to pathogen infection and high salinity conditions in transgenic tobacco plants Plant Mol. Biol. 55 61 81
Little, H.A., Papadopoulou, E., Hammar, S.A. & Grumet, R. 2007 The influence of ethylene perception on sex expression in transgenic melon as assessed by expression of the mutant ethylene receptor gene, At-etr1-1, under control of constitutive and floral-targeted promoters Sex. Plant Reprod. 20 123 136
Lund, S.T., Stall, R.E. & Klee, H.J. 1998 Ethylene regulates the susceptible response to pathogen infection in tomato Plant Cell 10 371 382
Martinex-Madrid, M.C., Flores, F. & Romojaro, F. 2002 Behavior of abscisic acid and polyamines in antisense ACC oxidase melon (Cucumis melo) during ripening Funct. Plant Biol. 29 865 872
McDonald, M.P. & Visser, E.J.W. 2003 A study of the interaction between auxin and ethylene in wild type and transgenic ethylene-insensitive tobacco during adventitious root formation induced by stagnant root zone conditions Plant Biol. 5 550 556
Mihaliak, C.A. 2002 Preparing and conducting field testing: An industry perspective Wolfenbarger L.L. Proceedings of a Workshop on Criteria for Field Testing of Plants with Engineered Regulatory, Metabolic, and Signaling Pathways 3–4 June 2002 Information Systems for Biotechnology Blacksburg, VA 57 61
Morgan, P.W. & Drew, M.C. 1997 Ethylene and plant responses to stress Physiol. Plant. 100 620 630
Nakajima, N., Itoh, T., Asai, N., Aona, M., Kubo, A., Azumi, Y., Kamada, H. & Saji, H. 2002 Improvement in ozone tolerance of tobacco plants with an antisense DNA for 1-aminocyclopropane-1-carboxylate synthase Plant Cell Environ. 25 727 735
Narumi, T., Aida, R., Ohmiya, A. & Satoh, S. 2005 Transformation of chrysanthemum with mutated ethylene receptor genes: mDG-ERS1 transgenes conferring reduced ethylene sensitivity and characterization of the transformants Postharvest Biol. Technol. 37 101 110
Nickson, T.E. 2008 Planning environmental risk assessment for genetically modified crops: Problem formulation for stress tolerant crops Plant Physiol. 147 494 502
Nukui, N., Ezura, H. & Minamisawa, K. 2004 Transgenic Lotus japonicus with an ethylene receptor gene Cm-ERS/H70A enhances formation of infection threads and nodule primordia Plant Cell Physiol. 45 427 435
Oeller, P.W., Lu, M.W., Taylor, L.P., Pike, D.A. & Theologis, A. 1991 Reversible inhibition of tomato fruit senescence by antisense RNA Science 254 437 439
Okubara, P.A. & Paulitz, T.C. 2005 Root defense responses to fungal pathogens: A molecular perspective Plant Soil 274 215 226
Papadopoulou, E. & Grumet, R. 2005 Brassinosteroid-induced femaleness in cucumber and relationship to ethylene production HortScience 40 1763 1767
Papadopoulou, E., Little, H.A., Hammar, S.A. & Grumet, R. 2005 Effect of modified endogenous ethylene production on sex expression, bisexual flower development and fruit set in melon (Cucumis melo L.) Sex. Plant Reprod. 18 131 142
Picton, S., Barton, S.L., Bouzayen, M., Hamilton, A.J. & Grierson, D. 1993 Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene forming enzyme transgene Plant J. 3 469 481
Pierek, R., Cuppens, M.L.C., Voesenek, L.A.C.J. & Visser, E.J.W. 2004 Interactions between ethylene and gibberellins in phytochrome-mediated shade avoidance responses in tobacco Plant Physiol. 136 2928 2936
Pierek, R., Visser, E.J.W., de Kroon, H. & Voesenek, L.A.C.J. 2003 Ethylene is required in tobacco to successfully compete with proximate neighbors Plant Cell Environ. 26 1229 1234
Pozo, M.J., Van Loon, L.C. & Pieterse, C.J.M. 2004 Jasmonates—Signals in plant–microbe interactions J. Plant Growth Regulat. 23 211 222
Rashotte, A.M., Chae, H.S., Maxwell, B.B. & Kieber, J.J. 2005 The interaction of cytokinin with other signals Physiol. Plant. 123 184 194
Reed, A.J., Magin, K.M., Anderson, J.S., Austin, G.D., Rangwala, T., Linde, D.C., Love, J.N., Rogers, S.G. & Fuchs, R.L. 1995 Delayed ripening tomato plants expressing the enzyme 1-aminocyclopropane-1-carboxylic acid deaminase. 1. Molecular characterization, enzyme expression, and fruit ripening traits J. Agr. Food Chem. 43 1954 1962
Robison, M.M., Shah, S., Tamot, B., Pauls, K.P., Moffatt, B.A. & Glicke, B.R. 2001 Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase Mol. Plant Pathol. 2 131 145
Rojo, E., Solano, R. & Sanchez-Serrano, J.J. 2003 Interactions between signaling compounds involved in plant defense J. Plant Growth Regulat. 22 82 98
Sahora, M.K., Sridhar, P. & Malik, V.S. 1998 Glyphosate-tolerant crops: Genes and enzymes J. Plant Biochem. Biotechnol. 7 65 72
Sasse, J.M. 2003 Physiological actions of brassionosteroids: An update J. Plant Growth Regulat. 22 276 288
Saxena, D. & Stotzky, G. 2001 Bt corn has a higher lignin content than non-Bt corn Amer. J. Bot. 88 1704 1706
Schaffer, R.J., Friel, E.N., Souleyre, E.J.F., Bolitho, K., Thoday, K., Ledger, S., Bowen, J.H., Ma, H., Nain, B., Cohen, D., Gleave, A.P., Crowhurst, R.N., Janssen, B.J., Yao, J.-L. & Newcomb, R.D. 2007 A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway Plant Physiol. 144 1899 1912
Shaw, J.F., Chen, H.H., Tsai, M.F., Kuo, C. & Huang, L.C. 2002 Extended flower longevity of Petunia hybrida plants transformed with boers, a mutated ERS gene Brassica oleracea Mol. Breed. 9 211 216
Shibuya, K., Barry, K.G., Ciardi, J.A., Loucas, H.M., Underwod, B.A., Nourizadeh, S., Exker, J.R., Klee, H.J. & Clark, D.J. 2004 The central role of PhEIN2 in ethylene responses throughout plant development in petunia Plant Physiol. 136 2900 2912
Silva, J.A., daCosta, T.S., Lucchetta, L., Marini, L.J., Zanuzo, M.R., Nora, L., Twyman, R.M. & Rombaldi, C.V. 2004 Characterization of ripening behavior in transgenic melons expressing and antisense 1-aminocyclopropane-1 carboxylate (ACC) oxidase gene from apple Postharvest Biol. Technol. 32 263 268
Sinn, J.P., Schlagnhaufer, C.D., Arteca, R.N. & Pell, E.J. 2004 Ozone-induced ethylene and foliar injury responses are altered in 1-aminocyclopropane-1-carboxylate synthase antisense potato plants New Phytol. 164 267 277
Sisler, E.C. & Serek, M. 1997 Inhibitors of ethylene responses in plants at the receptor level Physiol. Plant. 100 577 582
Snow, A.A., Andow, D.A., Gepts, P., Hallerman, E.M., Power, A., Tiedje, J.M. & Wolfenbarger, L.L. 2005 Genetically engineered organisms and the environment: Current status and recommendations Ecol. Appl. 15 377 404
Snow, A.A., Pilson, D., Rieseberg, L.H., Paulsen, M.J., Pleskac, N., Reagon, M.R., Wolf, D.E. & Selbow, S.M. 2003 A Bt transgene reduces herbivory and enhances fecundity in wild sunflowers. Ecol. Appl. 13 279 286
Sozzi, G.O., Fraschina, A.A. & Castro, M.A. 2001 Ripening-associated microstructural changes in antisense ACC synthase tomato fruit Food Sci. Technol. Intl. 7 59 71
Sriskandarajah, S., Mibus, H. & Serek, M. 2007 Transgenic Campanula carpatica plants with reduced ethylene sensitivity Plant Cell Rpt. 26 805 813
Stearns, J.C. & Glick, B.R. 2003 Transgenic plants with altered ethylene biosynthesis or perception Biotechnol. Adv. 21 193 210
Stearns, J.C., Shah, S., Greenberg, B.M., Dixon, D.G. & Glick, B.R. 2005 Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel Plant Physiol. Biochem. 43 701 708
Swarup, R., Parry, G., Graham, N., Allen, T. & Bennet, M. 2002 Auxin cross-talk: Integration of signaling pathways to control plant development Plant Mol. Biol. 49 411 426
Takada, K., Ishimaru, K., Minamisawa, K., Kamada, H. & Ezura, H. 2005 Expression of a mutated melon ethylene receptor gene, Cm-ETR1/H69A, affects stamen development in Nicotiana tabacum Plant Sci. 169 935 942
Trusov, Y. & Botella, J.R. 2006 Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.] J. Expt. Bot. 57 3953 3960
Vahala, J., Rounala, R., Keinanen, M., Tuominen, H. & Kangasjarvi, J. 2003 Ethylene insensitivity modulates ozone-induced cell death in birch Plant Physiol. 132 185 195
Van Zhong, G. & Burns, J.K. 2003 Profiling ethylene-related gene expression in Arabidopsis thaliana by microarray analysis Plant Mol. Biol. 53 117 131
Vardhini, B.V. & Rao, S. 2002 Acceleration of ripening of tomato pericarp discs by brassinosteroids Phytochemistry 16 843 847
Visser, E.J.W. & Voesenek, L.A.C.J. 2005 Acclimation to soil flooding—Sensing and signal transduction Plant Soil 274 197 214
Voesenek, L.A.C.J., Benschop, J.J., Bou, J., Cox, M.C.H., Groeneveld, H.W., Millenaar, F.F., Vreeburg, R.A.M. & Peeters, A.J.M. 2003 Interactions between plant hormones regulate submergence-induced shoot elongation in the flooding-tolerant dicot Rumex palustris Ann. Bot. (Lond.) 91 205 211
Wang, H., Huang, Z.J., Chen, Q., Zhang, Z.J., Zhang, H.B., Wu, Y.M., Huang, D.F. & Huang, R.F. 2004 Ectopic overexpression of tomato JERF3 in tobacco activates downstream gene expression and enhances salt tolerance Plant Mol. Biol. 55 183 192
Wang, K.L.C., Li, H. & Ecker, J.R. 2002 Ethylene biosynthesis and signaling networks Plant Cell 14 S131 S151
Whitelaw, C.A., Lyssenko, N.N., Chen, L., Zhou, D., Mattoo, A.K. & Tucker, M.L. 2002 Delayed abscission and shorter internodes correlate with a reduction in the ethylene receptor LeETR1 transcript in transgenic tomato Plant Physiol. 128 978 987
Wilkinson, J.Q., Lanahan, M.B., Clark, D.G., Bleecker, A.B., Chang, C., Meyerowitz, E.M. & Klee, H.J. 1997 A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants Nat. Biotechnol. 15 444 447
Woeste, K.E., Vogel, J.P. & Kieber, J.J. 1999 Factors regulating ethylene biosynthesis in etiolated Arabidopsis thaliana seedlings Physiol. Plant. 105 478 484
Wolfenbarger, L.L. & Grumet, R. 2002 Executive summary 5 12 Wolfenbarger L.L. Proceedings of a Workshop on Criteria for Field Testing of Plants with Engineered Regulatory, Metabolic, and Signaling Pathways 3–4 June 2002 Information Systems for Biotechnology Blacksburg, VA
Wong, W.S., Li, G.G., Ning, W., Xu, Z.F., Hsiao, W.L.W., Zhang, L.Y. & Li, N. 2001 Repression of chilling-induced ACC accumulation in transgenic citrus by over-production of antisense 1-aminocyloproplane-1-carboxylate synthase RNA Plant Sci. 161 969 977
Xiong, A.-S., Yao, Q.-H., Peng, R.-H., Li, X., Han, P.-L. & Fan, H.Q. 2005 Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato Plant Cell Rpt. 23 639 646
Yang, S.F. & Hoffman, N.E. 1984 Ethylene biosynthesis and its regulation in higher plants Annu. Rev. Plant Physiol. 35 155 189
Yang, S.F. & Oetiker, J.H. 1998 Molecular biology of ethylene biosynthesis and its application in horticulture J. Japan. Hort. Soc. 67 1209 1214
Yang, Z., Tian, L.N., Latoszek-Green, M., Brown, D. & Wu, K.Q. 2005 Arabidopsis ERF4 is a transcriptional repressor capable of modulating ethylene and abscisic acid responses Plant Mol. Biol. 58 585 596
Zhang, H.B., Zhang, D.B., Chen, J., Yang, H.D., Huang, Z.L., Huang, D.F., Wang, X.C. & Huang, R.F. 2004 Tomato stress-responsive factor TERF1 interacts with ethylene responsive element GCC box and regulates pathogen resistance to Ralstonia solanacearum Plant Mol. Biol. 55 825 834
Zhang, S.L., Zhang, Z.J., Chen, J., Chen, Q., Wang, X.C. & Huang, R.F. 2005 Expressing TERF1 in tobacco enhances drought tolerance and abscisic acid sensitivity during seedling development Planta 222 494 501