Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: Activities: During the 2012 research year, we completed the analyses of the transcriptomics data obtained with the Affymetric wheat chip and crown RNA from untreated and cold acclimated winter wheat lines varying in freeze survival. The lines were SD16029 (FR, 75 % freeze survival in the field) and SD16169 (FS, 30 % freeze survival in the field). We correlated the gene ontology terms for biological processes with cultivar freeze survival. Funding for this project allowed for the mentoring of 4 undergraduates and one Ph.D graduate student. The Ph.D student was successful with the project and is currently a postdoctoral fellow at the University of Wisconsin-Milwaukee. The most significant outcome was the identification of critical pathways that correlate with freeze survival. These findings have been disseminated at meetings as well as in peer-reviewed publications. Other outcomes include the generation of several datasets of wheat cold regulated genes; the identification of biological pathways that reflect how plants respond to low non-freezing temperature and pathways that may help explain freeze survival of winter wheat. The datasets have been deposited at the plant expression database "http://www.plexdb.org/ website accession number TA22" and the GEO database accession # GSE14697. These activities have also fostered the continued collaboration with Dr. Horvath at USDA ND. PARTICIPANTS: This project allowed for the training of undergraduate researchers as well as a Ph.D student. TARGET AUDIENCES: The target audiences were mainly other research groups studying abiotic stress, plant biology and crop breeding. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our results resulted in a change in knowledge reflected by new information significant enough to be included in publications. Three critical pathways identified were DREB2A, E2F and Rubisco pathways. This was a significant discovery because the DREB2A pathway is upregulated while both the E2F and Rubisco pathways are down regulated. We have not yet determined which if any of the three pathways control the other. However we hypothesize that the E2F pathway is the most critical of all three because of its role in controlling the cell cycle.
Publications
- Karki, A., Ge, X., Chen, D., and Sutton, F. 2011. Sparse Principal Component Analysis (SPCA) of Wheat Microarray data identifies co-expressed genes differentially regulated by cold acclimation. The Joint Statistical Meeting, 2011. Page 2159 - 2170.
- Karki A, Horvath D, Sutton F., Induction of DREB2A pathway with repression of E2F, jasmonic acid biosynthetic and photosynthesis pathways in cold acclimation-specific freeze-resistant wheat crown. Funct Integr Genomics (2013) 13: 57-65. DOI 10.1007/s10142-012-0303-2.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities: During the 2011 research year, we further examined the data generated from the wheat transcriptomics studies using the Affymetric wheat chip with crown RNA from untreated and cold acclimated winter wheat lines varying in freeze survival. The lines are SD16029 (FR, 75 % freeze survival in the field) and SD16169 (FS, 30 % freeze survival in the field). We used the statistical method, sparse principal component analysis (SPCA), to reduce the dimensionality of the dataset. We performed homology searches against the Arabidopsis database with our wheat sequences to obtain the Arabidopsis orthologues. We initiated collaboration with Dr. Horvath at USDA ND to identify the pathways reflected from the differentially expressed genes. We identified the gene ontology terms for biological processes, cellular components and molecular functions using the software Pathway Studio and are currently comparing those results with that obtained from software packages GOSTAT and PANTHER. Conditions were determined for production of sterile calli from embryos extracted from seeds of FR, FS and wild type lines. Freezing parameters were determined. Osmolyte leakage was standardized to measure cell survival. RNA isolation procedures are being finalized for maximum yield and for realtime RT_PCR for examination of the expression of the CBFs identified in our earlier studies as associated with freeze survival. The results from the calli studies will be compared with that published for the crown tissue. Activities also included validation of genes identified by the microarray analysis as associated with freeze survival. The transcription factors CBF- 22 and -2 appear to control the expression of the majority of abscisic acid dependent and independent cold-responsive genes (COR). Both sets of genes displayed the same patterns of expression in response to cold in both FR and FS lines. However, CBF-3, -5, -6, -12, -14, and -19 are significantly up-regulated in response to cold acclimation and differentially regulated between FR and FS lines. To date the target genes for this latter group of transcription factors have not been verified. From the expression patterns across the microarray, one candidate target gene was identified. This target gene COR518 displayed the same cold regulated expression pattern as the transcription factor CBF12. RT_PCR with crown RNA from both untreated and cold acclimated FR and FS lines was performed to verify the microarray results. Products: These activities have led to: the generation of several datasets of wheat cold regulated genes; the identification of biological pathways that reflect how plants respond to low non-freezing temperature and pathways that may help explain freeze survival of winter wheat. These activities have also fostered the continued collaboration with Dr. Horvath at USDA ND. The embryonic calli experiments are leading to the development of new tools for examination of freeze survival of hard red winter wheat. Two undergraduate and graduate students were involved with these activities. Dissemination: Our outreach activities included attendance at various symposia and conferences. PARTICIPANTS: During this period, two undergraduate and graduate students received research experience by their significant particiation in the activities. The PI played the significant role of supervising all participants. We collaborated with Dr. Horvath at the USDA ND with the pathway analysis aspect of the research and Drs Chen and Ge with the statistical analyses. TARGET AUDIENCES: Our research was presented at several bioinformatic scientific symposia. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The SPCA statistical tool used can be very valuable to reduction of the datasets used in microarray analyses as presented in our publication Karki et al (2011). We are the first to identify COR 518 as a target gene for CBF12. These findings are in preparation for submission. The wheat callus FR and FS experimental systems will be invaluable as we further test the roles of the CBFs and their target genes.
Publications
- Publications Karki, A., Ge, X., Chen, D., and Sutton, F. 2011. Sparse Principal Component Analysis (SPCA) of Wheat Microarray data identifies co-expressed genes differentially regulated by cold acclimation. The Joint Statistical Meeting, 2011. Page 2159 - 2170.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: OUTPUTS: The results published by Sutton et al., 2009 clearly demonstrated the link between expression of six transcription factors of the CBF class that are differentially regulated between cold acclimated freeze resistant Winoka mutant SD16029 (FR) and cold-acclimated freeze susceptible Winoka mutant SD16169 (FS). The output for the research period 2010 focused on identification of regulatory elements (RE) in the 5' regulatory region of the FR gene for the transcription factor CBF12 and determining whether these elements differed from that found in the Wild type Winoka CBF12 regulatory region. We successfully subcloned 1 kb fragments upstream of the transcription start site for both FR and WT CBF12 genes. Since we are working with a hexaploid wheat, it was necessary to determine for which of the A, B and D genomes did we have the 5'regulatory regions. This was determined by subcloning the 5' regions isolated from wild type genomic DNA, sequencing the regions and performing CLUSTALw analysis. The fragments were then grouped into three clusters. The Wild type sequences were then compared with the sequences of the subcloned FR 5'regulatory regions. We have identified a RE which differs by one nucleotide between the FR and WT 5'regulatory region. A comparison of this sequence with regulatory database elements has not allowed us to identify the transcription factor that would interact with this element. Our future plans are to link the regulatory regions containing the element to a reporter gene and determine whether it controls regulation by cold acclimation as observed for FR and WT wheat genotypes. PARTICIPANTS: One PhD graduate student and one undergraduate worked with the PI on this project. The PhD student had the opportunity to attend the ASPB International meeting that was held in Montreal Canada and presented the poster. TARGET AUDIENCES: The research is relevant to all groups who are interested in understanding the molecular basis of abiotic stress. The genes identified could be of great value to the plant breeders. Therefore the research is also relevant to plant breeders. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
IMPACT: Our results suggest that there may be a novel transcription factor that may be involved in conferring freeze survival of winter wheat. The transcription factor since it will be the controller of the CBF transcription factors will be the first "regulator of regulators" clearly identified for control of hexaploid winter wheat abiotic stress.
Publications
- Fedora Sutton and Amrit Karki (2010) Regulation of transcription factors linked to cold acclimation and freeze survival of hexaploid wheat. South Dakota Academy of Sciences Proceedings. Sutton Fedora (2010) Transcriptional Control of Gene Networks in Plant Systems. Oral presentation University of the West indies CaveHill Barbados.
- Karki A, Kontz B, Sutton F (2010)Regulation of Wheat Cbf12 as it relates to ploidy and cold acclimation. ASPB 2010 Abs # P07063 Montreal Canada
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We have a wheat experimental system comprising two Triticum aestivum lines cv"Winoka" that vary in field freeze survival. SD16029 with 75% survival was designated FR and SD160169 with 30% survival was designated FS. We have transcriptome data from cold acclimation of FR and FS lines(Sutton et al 2009). As we examined the data, we observed that there was a pattern of gene expression that was different not only between FR and FS but also among the wheat A,B and D genomes in response to cold acclimation. The expression of the Cbfs relative to the AB and D genomes was as follows: Cbf-10 on the B genome appeared to be not expressed or modulated by cold acclimation in either FR and FS. Yet Cbf-22 on the B genome was at least 4 fold up-regulated in FR and FS, although not differentially regulated between FR and FS. Cbf-B12 behaved like Cbf-B22 in FR in that it was highly up-regulated with cold acclimation whereas it behaved like Cbf-B10 in FS in that it appeared to be inactive on the B genome. The lack of expression on the B genome of FR was not particular to Cbf-B10, since Cbf-D22 showed a similar lack of expression compared to its homeolog on genome A and B. It is clear that all of the genomes are capable of expressing one or more of these Cbf genes. Except for Cbf-D22 which is silent, we are unable to provide information regarding the contributions to the microarray expression data for homeologs from the A and D genome for Cbf12, -14 or -3. If Cbf genes 3, 6, 12, 14 and 19 are necessary to confer freeze survival and if they are only expressed by one genome, it can be assumed that lack of expression of the single copy would have a significant impact on freeze survival. When all 5 of these genes are down-regulated with cold acclimation the plants were found to be freeze susceptible (FS). This down regulation depicted in the microarray data was confirmed by real-time RT-PCR. To determine whether the protein CBF12 from FR and FS was functional, the coding region of CBF12 was sub-cloned and sequenced. This was achieved by using Cbf12-ORF specific primers and PCR amplification of the genomic DNA of FR and FS. The resulting amplicons were subcloned into the pTOPO-ENTR vector and transformed into DH5α cells. Colonies selected on kanamycin plates were tested for inserts by PCR amplification with the Cbf12 primers. The deduced amino acid sequences for the Cbf12-Orf homeologs cloned from FR and FS were identical . What was particularly informative was the absence of any mutations of the AP2 domain which is the DNA binding domain of the CBFs. We still need to determine which of the homeologs are actively expressing CBF12 and verify that the transcripts from those homeologs are full length and unmodified. However, assuming that this preliminary data reflects the status of the authentic CBF12 proteins from FR and FS, we can assume that when Cbf12 is expressed as it is in the FR crown tissue, the resulting CBF12 protein will be functional. Results from this research were presented at the Annual Association of Plant Biology Meeting. PARTICIPANTS: Two graduate students, two undergraduates and the PI worked on this project. This was an excellent opportunity for the undergraduates to be involved in research at all levels. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our results suggest that there should be a change in the manner in which gene expression is described or considered in hexaploid wheat. As scientists discuss silencing genes in wheat, the following questions should be asked of them "Do you know which gene on which of the three genomes is active Which of these do you propose to silence and How will you ensure that you are silencing the appropriate homeolog". Our results suggest that as has been observed in other polyploids, one of the copies of a particular gene could serve to repress expression of its homeolog. Thus we need to expand our knowledge of gene expression in polyploids.
Publications
- Sutton F,Chen DG,Ge X,Kenefick D.(2009)Cbf genes of theFr-2 allele are differentially regulated between long-term cold acclimated crown tissue of freeze-resistantand-susceptible, winter wheat mutant lines. BMC PlantBiol.Mar 23;9:34.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In collaboration with Din Chen of the SDSU Mathematics department, Affymetric wheat chips consisting each of 52,000 probes were hybridized with cDNA derived from RNA isolated from crown tissue of Winter Wheat cultivars varying in freeze survival. The two cultivars are denoted FR and FS. FR refers to SD16029, 75% freeze survival Casselton ND and FS refers to SD 16169, 30% freeze survival. These cultivars were derived from azide mutagenesis of the winter wheat cultivar "Winoka". We have determined that there are three genes belonging to the central cluster of the (freeze resistance) Fr loci on the long arm of chromosome 5 that are differentially regulated between FR and FS lines. These three genes Cbf12, -14 and -19 are therefore candidates for major controllers of freeze survival of winter wheat. The other genes that were differentially regulated are believed to be down stream from the central cluster genes. We have also determined that a number of the cold-regulated genes are most likely under the control of the genes of the vernalization loci and not the Fr loci. Given that the two cultivars were derived by azide mutagenesis we must assume that the mutagenesis affected a transcription factor that controls the CBFs. Since freeze survival increased in the FR mutant, we are proposing that there is a gain of function mutation resulting in the de-repression of the key Cbf genes. The results from this study have been submitted for publication and have been presented as initial data in a Plant Genome proposal submitted to the NSF on January 21 st. PARTICIPANTS: Din Chen PhD, Xijin Ge PhD - Both are faculty in the Department of Mathematics and Statistics and worked on the analysis of the data. Sean Franklin- Undergraduate , worked with PI on the real time RT-PCR verification of the microarray data. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: It has previously been proposed by other researchers that differential expression of cold-regulated (Cor/Lea) genes are associated with freezing resistance. Our data provides new information relating the control of the Cor genes to the Vrn genes. This new information is based on our experimental system of two winter wheat genotypes varying in freeze survival. We have also removed from the list of candidate freeze resistant associated genes other Cbf genes for example Cbf-2.
Publications
- No publications reported this period
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