Progress 10/01/02 to 09/30/05
Outputs
Conversion of environmental signals into the appropriate cellular responses, whether these responses are changes in enzyme activity or alterations in gene expression, is important for survival of all organisms. In all eukaryotes, calcium is an important cellular second messenger. Calcium fluxes in the cytosol are a common mechanism for transmitting biological information and these fluxes must be detected and then properly decoded for the correct response to be initiated. In plants, a major group of calcium sensors are the calcium-dependent protein kinases (CDPKs). CDPKs have been identified throughout the plant kingdom, from single-celled algae to crop plants, and understanding their role in signal transduction is an active area of research. My laboratory has focused on characterizing CDPKs in the model plant Arabidopsis thaliana (mouse-eared cress). The goal of the current project was to elucidate how levels of CDPK transcripts, representing the initial step in gene
expression, differed between different plant organs and upon stress treatment. Semi-quantitative RT-PCR (reverse transcription-polymerase chain reaction) was used to measure gene expression. This technique allows comparison of relative RNA levels from different tissues, organs. To study expression under standard growth conditions, RNA was isolated from 11 different tissues, organs, or developmental stages. Primer pairs specific for 21 of the 34 Arabidopsis CDPKs were designed and tested to determine the optimum PCR cycle number that should be used for quantitation. EF1 (elongation factor 1) was included as an internal control since its expression is relatively unchanged in different tissues. We found that most of the 21 CDPKs that were tested were expressed in either 14-day old seedlings or in roots, although expression of two of the genes could only be detected in open flowers. Our results correlated well with several recent publications on global gene expression profiling in
Arabidopsis roots and flowers. At the start of this project, little was known about CDPK gene expression in Arabidopsis and few resources were available to researchers. During the course of our experiments, microarrays containing most of the Arabidopsis genes became available as research tools for conducting global studies on transcriptional profiling, although cost prohibited their use in our studies. However, data from many microarray experiments has been collected and is available for searching online. We conducted an in silico analysis of Arabidopsis gene expression and found that at least nine Arabidopsis genes are predominantly expressed in stamens, most likely in pollen. The remaining CDPK genes are expressed in most organs but are poorly expressed in stamens. In addition, about half of the Arabidopsis CDPK genes are highly expressed in roots. These data are consistent with our RT-PCR experiments and help to provide a more complete picture of CDPK gene expression in
Arabidopsis.
Impacts
Calcium-dependent protein kinases (CDPKs) are important for correct plant responses to drought stress, pathogen attack, wounding etc. Identifying where and when plants express CDPK genes is critical for understanding how these proteins participate in signal transduction. The results of this research can be applied to future studies on crop plants.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Calcium-dependent protein kinases (CDPK) are the primary enzymes in plants that respond to fluctuations in cytosolic calcium levels. CDPKs have been implicated in responses to a variety of environmental stresses such as pathogen attack, drought stress, and wounding. In all plants, there appear to be multiple genes that encode CDPKs. For instance, 34 genes for CDPKs have been identified in the fully-sequenced genome of the model plant Arabidopsis thaliana, while rice has at least 26 genes and potato at least four. At present, there is litte known about the functions of individual CDPKs. Due to the large size of this gene family, we and others have hypothesized that CDPKs are specialized for specific functions and therefore may have expression patterns that differ temporally, spatially, or in response to stress signals. As part of this work, we will generate data on expression of CDPK genes in Arabidopsis under standard growth conditions and after stress treatment. This
information will help us to make predictions and design future experiments to elucidate CDPK functions. Semi-quantitative RT-PCR (reverse transcription-polymerase chain reaction) will be used to measure gene expression. This technique allows comparison of relative RNA levels from different tissues, organs, or developmental stages or during a time course following a stress treatment. To study expression under standard growth conditions, RNA has been isolated from 11 different tissues, organs, or developmental stages. For stress-induced expression, plants are subjected to a stress treatment and RNA is isolated at various time points following the treatment. To date, we have isolated RNA from two different stress treatments. For RT-PCR, primer pairs specific for each CDPK must be designed and tested, after which the optimum number of PCR cycles must be determined. Determining the correct cycle number is a critical and time-consuming part of the experimental design, since product
accumulation will become saturated if too many cycles are used, while the product will not be visible if the PCR is performed for too few cycles. Currently, we have determined the optimum cycle number for 20 of the 29 CDPK genes that are known to be expressed. Our results to date clearly indicate that some CDPKs have distinct expression patterns while others are expressed at comparable levels in all tissues. In all experiments, EF1 (elongation factor 1) has been included as an internal control since its expression is relatively unchanged in different tissues. Future experiments will focus on determining the expression patterns and stress induction for additional CDPKs.
Impacts
Calcium-dependent protein kinases are known to be important for plant responses to pathogen attack, drought stress, wounding, etc. This goal of this research is to identify the specific CDPKs involved plant stress responses and to provide information on expression of CDPKs in different plant organs or tissues. In the future, it is possible that modification of the expression of CDPK genes could be used to adapt plants to specific growth conditions or to improve plant growth and productivity.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Thirty-four genes for calcium-dependent protein kinases (CDPKs) have been identified in the fully-sequenced genome of the model plant Arabidopsis thaliana, although only 29 of these genes are known to be expressed. CDPKs are the primary calcium-regulated protein kinase in plants and are implicated in responses to a variety of environmental stresses such as pathogen attack, drought stress, and wounding. At present, there is almost no data on the functions of individual CDPKs. As part of this proposal, we will generate data on both tissue-specific expression and stress-induced expression of these genes. This information will help us to make predictions and design future experiments in order to elucidate CDPK functions. Semi-quantitative RT-PCR (reverse transcription-polymerase chain reaction) is being used to measure gene expression. For tissue-specific expression studies, RNA has been isolated from 11 different tissue types or developmental stages. For stress-induced
expression, plants are subjected to a stress treatment and RNA is isolated at various time points following the treatment. To date, we have isolated RNA from two different stress treatments. The semi-quantitative RT-PCR technique will allow comparison of relative RNA levels, either between the different tissue samples or between time points following the stress treatment. In preparation for RT-PCR, primer pairs specific for each CDPK must be designed and tested, after which the optimum number of PCR cycles must be determined. Determining the correct cycle number is a critical (and time-consuming) part of the experimental design, since product accumulation will become saturated if too many cycles are used, while the product will not be visible if the PCR is performed for too few cycles. To date we have determined the optimum cycle number for 19 of the 29 expressed CDPK genes. Preliminary RT-PCR experiments with some of the CDPK primer pairs indicate that, as expected, expression levels
are not the same for all tissues; however these experiments have not been repeated enough times to allow us to draw final conclusions about expression patterns. Control primers were used to amplify EF1 (elongation factor 1), a gene whose expression is relatively stable in all tissues throughout plant development. As predicted, the levels of EF1 expression show only slight variability between tissues, indicating that the same amounts of RNA were used in all reactions.
Impacts
Calcium-dependent protein kinases are known to be important for plant responses to pathogen attack, drought stress, wounding, etc. This research has the potential to identify the specific CDPKs involved in the response to each stress so that the correct CDPK can be modified to adapt plants for specific environments.
Publications
- No publications reported this period
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