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William R. Woodson

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William R. Woodson

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.

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William R. Woodson

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William R. Woodson

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.

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William R. Woodson

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William R. Woodson

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Kanogwan Kerdnaimongkol and William R. Woodson

Transgenic tomatoes (Lycopersicon esculentum Mill. `Ohio 8245') expressing an antisense catalase gene (ASTOMCAT1) were used to test the hypothesis that modification of the reactive oxygen species scavenging mechanism in plants can lead to changes in oxidative stress tolerance. A 2- to 8-fold reduction in total catalase activity was detected in the leaf extracts of transformants. A 2-fold increase in levels of H2O2 was observed in the transgenic plants with reduced catalase activity. Electrophoretic characterization of multiple catalase isoforms revealed the specific suppression of CAT1 in transgenic plants. Homozygous plants carrying the antisense catalase transgene were used to study the effect of alteration in the expression of catalase on stress tolerance. Transgenic plants treated with 3% H2O2 showed visible damage within 24 hours and subsequently died. In contrast, wild-type and azygous control plants recovered from the treatment. Transgenic plants did not survive 4 °C chilling stress compared to control wild-type and azygous lines. Physiological analysis of these plants indicated that suppression of catalase activity in transgenic tomato led to enhanced sensitivity to oxidative stress. Our data support a role for catalase in oxidative stress defense system in tomato.

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Sven Verlinden and William R. Woodson

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.

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Sven Verlinden and William R. Woodson

High-temperature treatments can be used for disinfestation of a variety of horticultural crops. Carnation flowers were subjected to a heat treatment in order to determine if it is a viable option for disinfestation of this crop. Flowers were exposed to 45°C for 24 hr in the dark, while control flowers were held at RT for 24 hr in the dark. Subsequently, the flowers were held at RT in the light and monitored for ethylene production, an indicator of imminent floral senescence. In the heat-treated flowers, the ethylene climacteric occurred at 96 hr after the heat treatment, a delay of 12 hr when compared to the control. Peak ethylene production was decreased by 25% to 30% in heat-treated flowers. Northern blot analysis of the ethylene biosynthetic pathway genes, ACC synthase, and ACC oxidase, showed that the expression of these genes is delayed by 8 to 16 hr in heat-treated flowers. This indicates that the delay and decrease in ethylene production is at least, in part, due to a delay or reduction in the expression of these genes. Further investigation revealed a decreased responsiveness of the petals to ethylene. Petals from heat-treated and control flowers were exposed to 1 ppm ethylene for 0, 0.5, 1, 2, 4, 6, 12, and 32 hr. The heat-treated petals again showed a delay and a decrease in maximum ethylene production after exposure to ethylene. A delay in expression of ACC synthase and ACC oxidase was also observed. The beneficial effects of exposing carnation flowers to high temperatures, a delay in ethylene production, and reduced responsiveness to ethylene, suggest that heat treatments could be used for disinfestation of this crop.

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Amanda S. Brandt and William R. Woodson

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).