The target of many genetic engineering experiments is to inhibit the expression of an endogenous gene. For example, research in my laboratory attempts to suppress the expression of ethylene biosynthetic pathway genes to inhibit the production of ethylene and delay flower senescence. The silencing of endogenous genes is generally accomplished by engineering plants to express either antisense or sense RNAs homologous to the target sequence. The mechanism by which gene silencing occurs is not clearly understood. Genetic and molecular analyses of transgene-induced silencing has revealed both meiotically reversible and fully stable phenotypes resulting from the expression of the transgene. In several cases, the mechanisms potentially involved in the silencing of the transgene and concomitant reversion of phenotype have been studied. These include transgene copy number, configuration of the integrated DNA, level of transgene RNA, and environmental factors. In many cases the silencing of transgenes was correlated with DNA methylation. These phenomena and the implications for engineering horticultural crops to express transgenes will be discussed in this workshop.
The senescence of carnation (Dianthus caryophylus) flower petals is regulated by the phytohormone ethylene and is associated with the expression of a number of senescence-related genes. These genes encode enzymes in the ethylene biosynthetic pathway, including both ACC synthase and ACC oxidase. Members of these gene families are differentially regulated in floral organs, with specific members responsible for the increase in ethylene biosynthesis that leads to petal senescence. Pollination often serves as the external signal to initiate the senescence cascade. Following pollination, a rapid increase in ethylene production by the pistal occurs, which is subsequently followed by increased ethylene in the petal. This response is mediated by pollen–pistil interaction(s) that occurs only in compatible pollinations. Recent data indicate that the signal transduction cascade following this cell-cell communication involves protein phosphorylation, as pollination-induced ethylene is sensitive to protein kinase and phosphatase inhibitors. To date, our lab has cloned and characterized a number of senescence-related genes that are believed to play a role in the process of senescence. These include genes that encode enzymes involved in cell wall dissolution (b-galactosidase), protein degradation (cysteine proteinase) and detoxification of breakdown products (glutathione s-transferase). Many of these senescence-related genes are under the transcriptional regulation of ethylene, which has been characterized at the molecular level. A number of biotechnology approaches to controlling the senescence of flowers have been explored. These include the down-regulation of ethylene biosynthetic genes, the expression of a dominant-negative mutation of the ethylene receptor gene, and the expression of genes that lead to increased cytokinin levels in tissues. These will be discussed in relation to the potential for delaying senescence through genetic engineering.
The opening of Freesia hybrida Bailey flowers cut in the tight bud stage was promoted by treatment with sucrose and 200 mg·liter−1 8-hydroxyquinoline citrate. A pulse treatment for 24 to 48 hr with 20% sucrose resulted in complete inflorescence development and prolonged vase life. Reduced sucrose concentrations or increased pulse durations were not as effective. Pulse-treating flowers with 20% sucrose for 24 hr prior to 3 days of simulated shipping improved subsequent flower opening and vase life.
Ethylene plays a key regulatory role in carnation flower senescence. Flower senescence is associated with a significant increase in ethylene production. Continued perception of this ethylene by the flower is necessary to sustain the climacteric rise in ethylene and the expression of senescence related genes associated with senescence. In addition, increased sensitivity by the flower to ethylene during development and senescence has been observed. In order to study the perception of ethylene at the molecular level, an ethylene receptor gene was cloned from carnation petals. The clone, CARETR, shows 68% homology at the nucleic acid level with the Arabidopsis ethylene receptor gene, ETR1. Northern blot analysis revealed that CARETR is present as a low abundant transcript in petals, styles, and ovaries. Further analysis also showed that CARETR is upregulated during flower senescence. Treatment with the ethylene action inhibitor norbornadiene (NBD) resulted in decreased levels of CARETR transcripts. These data suggest that CARETR plays a role in the increased sensitivity of carnation flowers to ethylene during flower development and is involved in staging the rapid and orchestrated death of the flower.
In Dianthus caryophyllus flowers the pollinated stigma gives rise to signals that are translocated throughout the flower and ultimately result in corolla senescence. Pollination leads to a rapid increase in ethylene production by the pollinated styles followed by ethylene biosynthesis from the ovaries, the receptacle tissue, and lastly the petals. The accumulation of ACC in these floral tissues also correlates with the sequential pattern of ethylene production. Ethylene production by the pollinated style can be defined temporally by three distinct peaks, with the first peak detected as early as 1 hour after pollination. In a carnation flower with multiple styles it is also possible to detect ethylene production from an unpollinated style on a pollinated gynoecium by 1 hour after pollination. This finding provides evidence for very rapid post-pollination signaling between styles. ACC synthase expression is induced in pollinated styles as early as 1 hour after pollination, but no message is detected in pollinated ovaries. ACC synthase enzyme activity is also absent in the pollinated ovaries despite the accumulation of large amounts of ACC in the ovary after pollination. This indicates that ACC must be translocated between organs after pollination. When a pollinated styles is removed from the flower at least 12 hours after pollination the corolla will still senesce. This indicates that the pollination signal has exited the style by this time. Evidence in carnations suggests that ACC and ethylene may both be involved in aspects of post-pollination signaling.
We have investigated the patterns of ethylene biosynthesis in carnation (Dianthus caryophyllus L.) genotypes that exhibit extended vase life in comparison to flowers of White Sim'. `White Sim' flowers exhibited typical symptoms of senescence, including petal in-rolling and rapid wilting, beginning 5 days after harvest. In contrast, the other genotypes studied did not show petal in-rolling or rapid wilting associated with petal senescence. The first visible symptom of senescence in these flowers was necrosis of the petal tips, and it occurred from 3 to 7 days after the initial symptoms of senescence were seen in `White Sim' flowers. In all cases, the extended-vase-life genotypes did not exhibit the dramatic increase in ethylene production that typically accompanies petal senescence in carnation. This appeared to be the result of limited accumulation of ACC. In addition, flowers of these genotypes had limited capacity to convert ACC to ethylene. Therefore, we conclude that the low level of ethylene produced by these flowers during postharvest aging is the result of low activities of both ACC synthase and the ethylene-forming enzyme. Treatment of `White Sim' flowers at anthesis with 1.0 μl ethylene/liter resulted in the induction of increased ethylene biosynthesis and premature petal senescence. The extended-vase-life genotypes exhibited varying responses to ethylene treatment. One genotype (87-37G-2) produced elevated ethylene and senesced prematurely, as did flowers of `White Sim'. A second genotype (82-1) was induced to senesce by ethylene treatment but did not produce increased ethylene. A third genotype (799) was unaffected by ethylene treatment. The results of this study suggest these extended-vase-life genotypes are representative of genetic differences in the capacity to synthesize and respond to ethylene. Chemical name used: 1-aminocyclopropane-1-carboxylic acid (ACC).