ABSTRACT
The tobacco nuclear matrix attachment region (MAR), RB7, has been shown to have a much greater effect on transgene expression in cultured cells than in transgenic plants. This is comparable to work in mouse systems showing that MARs have a positive effect on transgene expression in embryonic tissues but not adult tissues. There are several possible explanations for these observations. One is that cell differentiation state and proliferation rate can affect MAR function. We tested this possibility by initiating suspension cell cultures from well-characterized transgenic plants transformed with 35S::GUS with and without flanking MARs and then comparing GUS specific activity in the cell lines to those of the transgenic plants from which the cell lines were derived. If cell differentiation state and proliferation rate do affect MAR function, we would expect the ratio of transgene expression (cell suspensions : plants) to be greater in MAR lines than in control lines. This turned out not to be the case. Thus, it appears that MAR function is not enhanced simply because cells in culture divide rapidly and are not differentiated. Because in animal systems the chromosomal protein HMG-I/Y has been shown to be upregulated in proliferating cells and may have a role in MAR function, we have also examined the levels of the tobacco HMG-I/Y homolog by immunoblotting. The level of this protein does not differ between primary transformant cultured cells (NT-1) and Nicotiana tabacum plants (SR-1). However, a higher molecular weight cross-reacting polypeptide was found in nuclei from the NT-1 cell suspensions but was not detected in SR-1 leaf nuclei or cell suspensions derived from the SR-1 plants.
Subject(s)
HMGA1a Protein/genetics , Nicotiana/genetics , Plants, Genetically Modified , Cell Differentiation , Cells, Cultured , Gene Expression , HMGA1a Protein/metabolism , Nicotiana/cytologySubject(s)
DNA, Recombinant/metabolism , Gene Expression , Nicotiana/metabolism , Nuclear Matrix/metabolism , Plants, Toxic , Transgenes , Biolistics , Cells, Cultured , Chromatin/metabolism , DNA, Recombinant/genetics , Gene Transfer Techniques , Genes, Reporter , Glucuronidase/biosynthesis , Glucuronidase/genetics , Nucleosomes/metabolism , Nicotiana/cytologyABSTRACT
Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant. A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue mannitol concentration are inversely related. MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.
Subject(s)
DNA, Plant/genetics , Mannitol Dehydrogenases/genetics , Plant Proteins/genetics , Amino Acid Sequence , Base Sequence , DNA, Complementary/genetics , Molecular Sequence Data , Plant Proteins/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , Salicylates/pharmacology , Salicylic Acid , Sequence Homology, Amino Acid , Vegetables/genetics , Vegetables/metabolismABSTRACT
Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-[2-3H] inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate.
