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Final Report: Genetically Engineered Potato Plants Which Do Not Produce O3-induced Ethylene A Mechanism of O3 Tolerance?
EPA Grant Number: R823193Title: Genetically Engineered Potato Plants Which Do Not Produce O3-induced Ethylene A Mechanism of O3 Tolerance?
Investigators: Pell, Eva J. , Arteca, Richard N.
Institution: Pennsylvania State University - Main Campus
EPA Project Officer: Manty, Dale
Project Period: October 1, 1995 through September 30, 1998
Project Amount: $412,615
RFA: Exploratory Research - Environmental Biology (1995)
Research Category: Biology/Life Sciences
Description:
Objective:Upon exposure to the air pollutant ozone (O3), many plants emit the gaseous phytohormone ethylene (C2H4). A number of researchers have suggested that O3-induced C2H4 may play a role in many of the subsequent effects of this pollutant.
The enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase is considered to be the rate-limiting enzyme in the pathway for C2H4 production. At the time of the project proposal, we had identified an ACC synthase gene (ST-ACS4), which is induced during O3 exposure. Our objectives were to: (1) produce transformed potato plants that carry the antisense for ST-ACS4, and (2) test whether suppression of ST-ACS4 expression results in lessened sensitivity to O3.
In subsequent experiments, a second O3-induced potato ACC synthase gene (ST-ACS5) was identified. Both genes are also expressed in response to other stresses including copper and the fungal pathogen, Alternaria solani. In response to each of these stresses, ST-ACS5 is expressed earlier than ST-ACS4. To more clearly elucidate the individual role of each of these genes in the O3 response of potato, we produced separate plant lines carrying the antisense for each gene. Initially, cultivar Norchip was to be used in this project; however, this cultivar proved recalcitrant to in vitro shoot regeneration. Cultivar FL1607, which has been used extensively by other researchers for Agrobacterium-mediated transformation, was subsequently used in the project.
Summary/Accomplishments (Outputs/Outcomes):Complementary DNA (cDNA) fragments of ST-ACS4 and ST-ACS5 were cloned separately in antisense orientation into a plant transformation vector under the control of the CaM 35S promoter and the nos terminator. The vector carries the neomycin phosphotransferase (NPT II) gene, which confers kanamycin resistance (Kr). The antisense constructs for ST-ACS4 and ST-ACS5 were called pCDS4A and pCDS5A, respectively. Vectors pCDS4A and pCDS5A were introduced into A. tumefaciens by electroporation and antisense orientation of the cloned genes was confirmed by restriction enzyme (RE) digest and agarose gel analysis.
In early trials, one hundred sixty putative transformants were recovered via a standard plant transformation protocol. These plants did not grow well on kanamycin media, nor did they demonstrate NPT II enzyme activity or show extra ACC synthase gene bands in Southern analysis. We subsequently switched to the transformation method used in the University of Wisconsin lab of S. Austin-Phillips, and were successful in generating Kr potato lines.
To ensure that each regenerated plant line represents a distinct transformation event, only a single shoot was selected from each leaf callus mass. Line name designations indicate the ACC synthase antisense construct being introduced (?4' for pCDS4A, ?5' for pCDS5A) and the A. tumefaciens strain with which plants were transformed (?L' for LBA4404, ?G' for GV3101). For example, line G5-2 is the second line recovered from transformation with A. tumefaciens strain GV3101 carrying pCDS5A. L4-18 is the 18th line recovered from transformation with A. tumefaciens strain LBA4404 carrying pCDS4A.
Sixty-six lines were recovered from transformations with pCDS4A, and 56 lines were recovered from transformation with pCDS5A. Lines that showed no NPT II activity or would not grow vigorously on kanamycin medium were not maintained. Southern analysis of 30 putative antisense lines showed that Kr was not a guarantee that the antisense insert of interest had been incorporated into the plant genome. Several Kr lines did not carry any extra copies of the ST-ACS gene, while other lines carried as many as six additional gene copies.
After receiving approval from USDA APHIS, a field study was established in 1999 with six antisense potato lines (G4-9, G4-18, G4-40, L5-3, L5-14, and L5-17). The objectives were to examine agronomic characteristics of transgenic lines, and to observe their visible response to chronic ozone under field conditions. Included in the field study were non-transformed FL1607 plants as a "control" line. At the time of planting, the ability of the transgenic lines to produce ethylene in response to O3 had not yet been determined.
A border crop of cultivar Chieftain was established to provide a buffer between experimental lines. After emergence of the border crop, 12 open-topped gas exclusion chambers were placed in the field. The area inside each chamber permitted the transplanting of 4 pairs of plants (a pair from each of 3 antisense lines and the control FL1607 line) separated by single border plants. Twelve days after transplanting, O3 exposures were initiated.
All chambers received charcoal-filtered (CF) air; six chambers (three per antisense gene) were supplemented with 80 ppb O3 between 1000 and 1800 hours every day from July 1 through September 3. On September 9, vines were removed and dried to constant weight. Tubers were hand-harvested, weighed and graded for size, then diced, frozen in liquid nitrogen and freeze-dried to constant weight.
Figure 1 shows the vine dry weight (dwt) values of the field-grown potato plants. Two general observations can be made when examining the field plant performance as illustrated in Figure 1. First, the most striking differences in plant productivity are due to line differences, not the O3 treatment. Second, the non-transformed FL1607 plants were the most strongly affected by the O3 treatment. Similar response trends were apparent for tuber fresh weight (fwt) and tuber number (data not shown). Examination of tuber fwt and percent dry matter by tuber size grades did not show any significant effects of transgene or treatment (data not shown).
Statistical significance of O3 treatment effects and gene insertion effects are shown in Table 1. Effects on field performance due to transformation alone can be seen by comparing CF air grown transgenic plants to CF air grown FL1607 (Table 1 A). Presence of inserted genes sometimes had a deleterious effect in the field. Two transgenic lines, G4-9 and L5-17, did not grow as vigorously as the other lines; both exhibited leaf rolling and premature vine decline. In CF air, these 2 lines had significantly lower vine dwt, produced fewer tubers, and had lower tuber fwt than the FL1607 plants. CF air, lines L5-3 and L5-14 also had lower vine dwt and L5-3 had lower tuber fwt than CF air FL1607, but the differences were less significant than those seen in lines G4-9 and L5-17.
Ozone injury symptoms appeared on leaves of the transplanted lines the first week in August. Lines L5-3, L5-14, G4-18 and G4-40 did not differ from the FL1607 plants in severity of visible O3 injury. On August 23, after 7 weeks of ozone exposure, these five lines all had mild to moderate stippling or bronzing of leaves on the lower 2/3rds of the canopy, with mild senescence on lower leaves. Lines G4-9 and L5-17, however, displayed severe O3 injury symptoms, with extensive stippling even on the youngest leaves. Foliage throughout the canopies of O3-treated G4-9 and L5-17 plants showed premature senescence and the oldest leaves were curled and necrotic.
When comparing biomass of transgenic lines grown under chronic O3 stress to their cohort O3-grown FL1607 plants, there are few statistically significant differences (Table 1 B). Tuber fwt of L5-17 and tuber dry matter of L5-3 and G4-40 were lower than that of similarly treated FL1607 plants.
When CF air- and O3-treated plants are compared within each line (Table 1 C), it can be observed that O3 only had a significant negative effect on FL1607. Ozone-grown FL1607 plants had significant reductions in vine dwt and in number of tubers produced. No significant effect of O3 was seen in the percent dry matter of tubers of any line.
None of the growth irregularities observed in the field were manifested when these lines were grown in a CF greenhouse. Other researchers working with transgenic potato have reported disparities between growth habits of field- and greenhouse-grown transgenic plants. These findings highlight the need for field testing of antisense lines before concluding that they are agronomically acceptable.
Kr lines were screened in the greenhouse for O3-induced C2H4 production. In vitro plants were transplanted to a CF greenhouse supplied with 400 uE ?2 s-1 of supplemental light for a 16 h day. Approximately 6 weeks after planting, when plants had about 12 leaves, they were given an acute O3 exposure. Plants were placed in continuous stirred tank reactors (CSTRs) supplied with CF air or CF air supplemented with 250 ppb O3 for 1.5 h. Non-transformed FL1607 plants were simultaneously subjected to the O3 and CF air treatments, to compare the responses of the non-transformed and transformed lines. Exposure of each line was replicated at least twice. At the conclusion of the O3 exposure, leaf 4 from the apex was frozen in liquid N2 for RNA analyses. Leaf 5 from the apex was collected for ethylene measurement.
Of 31 Kr lines tested, 10 lines consistently demonstrated at least a 25 percent reduction in C2H4 emission in response to O3 when compared to O3-treated FL1607 plants (Table 2). Another 9 transgenic lines produced at least 25 percent more C2H4 after acute O3 exposure than did the O3-treated FL1607 plants, while the C2H4 emission of remaining lines did not differ from that of non-transformed plants after acute O3 exposure. The presence of additional gene inserts of ST-ACS4 or ST-ACS5 did not guarantee that potato plants would be prevented from producing C2H4 in response to O3. As has been observed by other researchers, insertion of even multiple antisense gene copies is not always effective in suppressing the sense transcript. This may be especially true for multigene families, such as the ACC synthase genes. For reasons that are unclear, insertion of antisense genes can sometimes increase expression of the target gene. This may be occurring in some of our transgenic lines.
It is interesting to note that the lines with low vigor in the field (G4-9 and G5-17) are among the lines that produced more C2H4 in the greenhouse studies. It is possible that another stress in the field triggered an overproduction of C2H4 by these lines, since this reduced vigor was seen in CF air, and was therefore not a result of C2H4 overproduction due to O3 stress.
In previous studies, we observed that ST-ACS5 is expressed early during acute O3 exposure of potato (within 30 min), followed by expression of ST-ACS4 (within 1.5 hr). We speculated that expression of ST-ACS5 might be required for expression of ST-ACS4. In greenhouse experiments, lines that were transformed with pCDS5A were more likely to have reduced O3-induced C2H4 production than were the pCDS4A-transformed lines (Table 2). Seven of the 16 pCDS4A-transformed lines tested had reduced C2H4 emission, while only 3 of the 15 pCDS4A-transformed lines showed similar reductions in C2H4 emission. The only line demonstrating almost complete suppression of the O3 response is an ST-ACS5 antisense line (G5-2).
In contrast, more pCDS4A-transformed lines appear to overproduce C2H4 during acute exposures. Seven pCDS4A-transformed lines showed this tendency for C2H4 overproduction, while only 2 pCDS5A-transformed lines showed such a response (Table 2).
It appears that suppression of ST-ACS5 may be more effective in preventing O3-induced ethylene production than suppression of ST-ACS4 alone. Studies in which antisense for both O3-induced ACC synthase genes are inserted into potato plants might enable maximal suppression of the O3 response. Such studies were beyond the scope of this project.
Gene expression studies are being conducted with lines used in the field experiment and lines demonstrating reduced O3-induced C2H4 emission. Potato leaves collected during greenhouse O3 experiments are used in northern analysis. A positive control of RNA extracted from naturally senescing FL1607 leaves is included, to verify that we are able to detect all of the ACC synthase gene products of interest.
Single stranded RNA probes are made for six potato genes: ACC synthase genes 1,3,4, and 5, rbcS, and 18S ribosomal. By transcribing the non-coding strands of the genes, we are able to detect sense transcripts and avoid cross-hybridization with the antisense RNA in transformed plants. ACC synthase and rbcS levels are expressed as a percent of each sample's ribosomal signal. Linearity of the signal is verified by probing serial dilutions of in vitro transcribed sense RNA of ST-ACS4 and ST-ACS5.
The results of these studies will clarify whether insertion of antisense ST-ACS4 or ST-ACS5 effectively prevented expression of the target genes, and whether overexpression of the target genes is occurring in some transgenic lines. They also will elucidate how antisense suppression of one ACC synthase gene affects the expression of the other ACC synthase and rbcS genes with and without O3 stress. The results of the gene expression studies will be published in the coming year.
General Summary. When potato plants are treated with ozone they produce ethylene, a plant hormone that induces many responses including foliar aging. Several genes encode for an enzyme, ACC synthase, that regulates ethylene synthesis. We cloned two genes for this enzyme and produced potato plants that carried the antisense to these genes. We hypothesized that these genetically transformed plants would have reduced capacity to synthesize ethylene, and as a result, would exhibit fewer adverse responses to ozone. The work reported here demonstrates that it was possible to prevent O3-induced C2H4 production in potato through introduction of antisense DNA for the gene ST-ACS5, although this effect was complete in only one out of several tested transgenic lines. Introduction of antisense DNA for another, later-expressing ACC synthase gene, ST-ACS4, was not as effective at preventing the O3 response. This work also reinforces the assertion that agronomic performance of transgenic potato lines must be field verified. There is potential for the antisense genotypes to serve as a source for O3 tolerant germplasm, but further tests would be needed to test the efficacy and safety of such an approach.
Acknowledgments. Shoot tip cultures of FL1607 were a gift of A.K. Handa of Purdue University. Transformed kanamycin resistant (Kr) potato plants were received as a gift from S. Austin-Phillips of the University of Wisconsin, Madison, and were used as a benchmark in tests for verifying successful transformations of FL1607. We are indebted to S. Austin-Phillips for advice on A. tumefaciens?mediated transformation of potato.
Supplemental Keywords:ozone, tropospheric, effects, enzymes, agriculture, molecular biology, air pollution. , Scientific Discipline, Health, Biology, Risk Assessments, Chemistry, Environmental Chemistry, Engineering, Genetics, ethylene, ozone, biosynthesis, agrobacterium cell supression, genetic engineering, genetic analysis, germplasm, pyridoxal phosphate inhibitor
Progress and Final Reports:
1999 Progress Report
Original Abstract