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Ronald T. Nagao
An increase in temperature (e.g. 40 C treatment) causes soybean seedlings to stop normal protein synthesis and synthesize a limited number of HS proteins (HS response). In contrast to animal systems where the high molecular weight HS proteins predominate, in soybeans the low molecular weight (15 to 18, 21 to 24 and 26 to 28 kD) families of HS proteins are most abundant. Research involving high temperature stress (HS) and other environmental stress agents on gene expression emphasizes analysis of HS genes including gene structure and regulation. Previous research has shown that the synthesis of HS proteins correlates with thermotolerance, which is the capacity to withstand a severe (lethal) temperature treatment if previously given a preconditioning heat treatment. Treatment with arsenite and cadmium lead to HS gene expression; thermotolerance is also induced by arsenite but not by cadmium. Amino acid analogues induce HS gene transcription, but thermotolerance is not acquired. The physiological/biochemical role(s) of HS proteins in cellular function and the acquisition of thermotolerance is one area of study.
The study of the molecular mechanisms of the regulation of HS gene expression has used a number of different approaches. The use of short-term heat treatments and cyclical heat treatments have separated the induction of HS gene transcription from that of sustained synthesis. Run-off transcription studies indicate that HS gene transcription is rapidly activated following heat treatment, but that transcription slows within 1-2 hours even during continuous heat treatment, a phenomenon termed self-regulation. The steady state level of HS mRNAs isolated from seedlings after continuous heat treatment or following return to control temperatures indicates that HS mRNA is more stable at HS temperatures than at control temperatures. While HS mRNA appear to be selectively translated at HS temperatures, most control mRNAs appear to be stable during HS. The intriguing question of the mechanism of self-regulation and how HS and control temperature mRNAs are differentiated at various temperatures is a major area of investigation.
Some classes of HS proteins undergo selective localization to organelles during HS while other classes remain soluble. Characterization and quantitation of HS proteins during localization after various HS and recovery regimes may provide insight concerning the function of the various classes of HS proteins. Further studies using the techniques of immunocytochemistry at the light and electron microscope levels with antibodies raised to various classes of HS proteins are planned to further characterize localization and to possibly functionally differentiate classes of HS proteins.
Initial experiments demonstrate the efficacy of using a HS promoter cassette to regulate expression of a gene and indicate that this low molecular weight HS protein gene promoter is very much more active than the constitutively expressed CaMV 35S promoter. This is important bcause in principle any gene can be cloned into this cassette, transferred into plants, and be thermally regulated by this strong, inducible promoter. Additionally, while this HS promoter is very active at HS temperatures and has very low activity at control temperatures but significant activity can be expressed at moderately elevated temperatures without induction of the full HS response. Thus expression of a sequence can be achieved without the physiological and biochemical changes associated with a full HS response. A gene encoding an enzyme that catalyzes cytokinin biosynthesis has been cloned into the HS cassette; heat treatment of transgenic tobacco plants caused phenotypic changes suggestive of cytokinin production. Further studies to characterize this promoter system in transgenic plants, including identification of tissue or developmental specificity, if any, should help evaluate the general usefulness of this promoter system as a basic research tool or possibly for the inducible regulation of beneficial genes introduced into crop plants.
Research conducted on the hormonal control of growth and development has emphasized the isolation and characterization of auxin-regulated genes and their expression. Families of genes that are up-regulated or down-regulated following auxin treatment have been isolated. A developmentally regulated family of proline-rich cell wall protein genes (SbPRP-1, SbPRP-2, and SBPRP-3) has also been isolated. Localization of the mRNAs transcribed from the auxin-regulated genes (Aux28 and Aux22) and the mRNAs for the family of SbPRP genes by in situ hybridization and tissue print hybridization suggest organ- and cell-specific expression of these genes. Further experiments using in situ hybridization to examine the expression of these genes families in additional tissues and during cell/organ development and maturation may provide clues as to the function(s) of these gene products.
Additional experiments to help gain some understanding of the possible function of the auxin-regulated genes are being conducted in Arabidopsis where genes homologous to Aux28 and Aux22 (as determined by physiological responsiveness and sequence data) have been isolated. Promoter deletion constructs of the Arabidopsis auxin-regulated genes fused to a LacZ reporter gene have been constructed and transformed into Arabidopsis. Characterization of these promoter deletion constructs are in progress.
Analysis of potential cis-regulatory elements of the Aux28 and Aux22 genes of soybean is continuing using gel retardation and transient expression assays.
Using the HS promoter cassette, constructs have been made to conditionally underexpress and to overexpress the auxin-regulated protein coding sequences. The immediate goal of these experiments is to determine if there are any phenotypic changes in transgenic Arabidopsis plants that can be correlated with the overexpression or underexpression of the gene products of these auxin-regulated genes.
Ainley, W. M., K. J. McNeil, J. W. Hill, W. L. Lingle, R. B. Simpson, M. L. Brenner, R. T. Nagao and J. L. Key. 1993. Regulatable endogenous production of cytokinins up to toxic levels in transgenic plants and plant tissues. Plant Mol. Biol. 22: 13-23.
Datta, N., P. R. LaFayette, P. A. Kroner, R. T. Nagao and J. L. Key. 1993. Isolation and characterization of three families of auxin down-regulated cDNA clones. Plant Mol. Biol. 21: 859-869.
Nagao, R. T., J. A. Kimpel and J. L. Key. 1990. Molecular and cellular biology of the heat-shock response. In: Advances in Genetics, Vol. 28: 235-274.
Nagao, R. T., V. H. Goekjian, J. C. Hong and J. L. Key. 1993. Identification of protein-binding DNA sequences in an auxin-regulated gene of soybean. Plant Mol. Biol. 21: 1147-1162.
Wyatt, R. E., R. T. Nagao and J. L. Key. 1992. Patterns of soybean proline-rich protein gene expression. The Plant Cell 4: 99-110.
Wyatt, R. E., W. M. Ainley, R. T. Nagao, T. W. Conner and J. L. Key. 1993. Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgeic plants. Plant Mol. Biol. 22:731-749.
Environmental stress mediated changes in transcriptional and translational regulation of protein synthesis in crop plants. U.S. Department of Energy - Biological Energy Research Program. J. L. Key and R. T. Nagao. Co-PI s.
Genetic transformation of peanut. Gold Kist. W. Parrot, S. Merkle and R. T. Nagao. Co-PI s.
Ginger Goekjian, Research Coordinator, M.S., University of Georgia.
Lynn Hill, Research Technician, M.S. University of Georgia.
Yuh-Ru Julie Lee, Graduate Student (Ph.D.), B.S. National Taiwan University. Characterization and functional study of soybean heat shock proteins.
Kevin O Grady, Postdoctoral Research Associate, Ph.D. University of Florida, Gainesville, FL. Localization of heat shock proteins.