Progress 11/15/00 to 11/14/03
Outputs
The objective of this project was to test whether specific cell wall proteins and enzymes are necessary or sufficient for tomato (LYCOPERSICON ESCULENTUM) seed germination. Genes encoding cell wall hydrolases (e.g., endo-O-mannanase [LeMAN2] and xyloglucan endotransglycosylase [LeXET4]) and an expansin (LeEXP4) are expressed specifically in the endosperm tissue that surrounds the radicle tip prior to radicle emergence, and a different expansin gene (LeEXP8) is expressed in the cortex of the radicle at the same time. In addition, the regulatory subunit gene of the SnRK1 kinase complex (LeSNF4) is expressed during seed development and in dormant seeds, but disappears rapidly in germinating seeds, suggesting involvement in the regulation of reserve accumulation and mobilization. An inducible promoter (XVE system controlled by a steroid regulator, estradiol) was used to develop transgenic tomato plants in which each of these genes could be induced or suppressed by the
application of estradiol to determine the consequences for germination. Transgenic plants were developed for each of these five genes in sense and RNA suppression configurations as well as green fluorescent protein (GFP) fusions, all under the control of the inducible promoter. These first generation plants are being genetically and physiologically characterized to identify single inserts and to produce homozygous seeds for use in physiological experiments. Preliminary experiments with segregating seed populations were done to determine whether the inducible promoter was functional and whether the genes could be induced in intact seeds. Overexpression of target genes in response to estradiol can be detected in bisected seeds, indicating that the promoter is present and inducible in the transgenic seeds. However, estradiol apparently is unable to penetrate through the testa and outer endosperm tissues of tomato seeds, as no induction of gene expression could be detected in intact
seeds. Strategies to increase the permeability of the seeds to the inducer are being tested.
Impacts
Identification of the specific genes required for seed germination will clarify the mechanisms involved in this process. These mechanisms can then be targeted for modification to improve or regulate germination for agricultural purposes. The ability to control the expression of transgenes in seeds with a specific inducer would have many applications in seed biology and crop production.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The objective of this project is to test whether specific cell wall proteins and enzymes are necessary or sufficient for tomato (LYCOPERSICON ESCULENTUM) seed germination. Several genes encoding cell wall hydrolases (e.g., endo-B-mannanase [LeMAN2] and xyloglucan endotransglycosylase [LeXET4]) and an expansin (LeEXP4) are expressed specifically in the endosperm tissue that surrounds the radicle tip prior to radicle emergence. In addition, a different expansin gene (LeEXP8) is expressed in the cortex of the radicle at the same time. Genes of the SnRK1 kinase complex are also expressed in developing and germinating seeds, possibly being involved in the regulation of reserve accumulation and mobilization. In particular, the regulatory subunit gene of this complex (LeSNF4) is expressed during seed development and in dormant seeds, but disappears rapidly in germinating seeds. An inducible promoter (XVE system controlled by estradiol) was used to develop transgenic tomato
plants in which each of these genes can be induced or suppressed by the application of a steroid regulator. In this way, each of these hydrolases, expansins or kinase subunit can be either enhanced or repressed to determine the consequences for germination. In addition, vectors containing fusion proteins between the target proteins and the green fluorescent protein (GFP) were constructed and transformed into plants to allow the expression and cellular location of these proteins to be identified using fluorescence microscopy. The primary transgenic plants have been developed for each of these five genes in sense and RNA suppression configurations as well as GFP fusions, all under the control of the inducible promoter. These plants are being genetically and physiologically characterized to identify single inserts and to produce homozygous seeds for use in physiological experiments. Physiological experiments to test the roles of these genes in seed germination will be conducted in the
coming year using these transgenic plants. These plants will also be crossed to mutants deficient in abscisic acid or gibberellin synthesis to further define the hormonal regulation and functions of the genes.
Impacts
Identification of the specific genes required for seed germination will clarify the mechanisms involved in this process. These mechanisms can then be targeted for modification to improve or regulate germination for agricultural purposes. Insight will also be gained into how these proteins act in other processes as well, such as cell expansion and metabolic regulation.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The objective of this project is to test whether specific cell wall proteins and enzymes are essential for tomato (LYCOPERSICON ESCULENTUM) seed germination. Several genes encoding cell wall hydrolases (e.g., endo-b-mannanase, xyloglucan endotransglycosylase, polygalacturonase) and an expansin (LeEXP4) are expressed specifically in the endosperm tissue that surrounds the radicle tip prior to radicle emergence. In addition, a different expansin gene (LeEXP8) is expressed in the cortex of the radicle at the same time. An inducible promoter (XVE system) will be used to develop transgenic tomato plants in which each of these genes can be induced or suppressed by the application of a steroid regulator. In this way, each of these hydrolases or expansins can be either enhanced or repressed to determine the consequences for germination. In addition, constructs containing fusion proteins between the target proteins and the green fluorescent protein will also be transformed
into plants. This will allow the expression and cellular location of these proteins to be identified using fluorescence microscopy. The primary transgenic plants will also be crossed together to determine the consequences of altering the expression of more than one of these components simultaneously. They will also be crossed to mutants deficient in abscisic acid or gibberellin synthesis to further define their hormonal regulation and function. To date on this project, the transformation vectors have been constructed and are being transformed into tomato plants at this time. Transgenic plants are expected to be recovered and characterized during the coming year.
Impacts
Identification of the specific genes required for seed germination will clarify the mechanisms involved in this process. These mechanisms can then be targeted for modification to improve or regulate germination for agricultural purposes.
Publications
- No publications reported this period
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