Progress 08/01/07 to 07/31/11
Outputs
OUTPUTS: The temporal expression of microRNA398 and two of its' targets genes (superoxide dsimutases, CSD1 and CSD2) has been analyzed in leaves, roots, stem, inflorescence as well as in seedlings and adult plants of tomato and rice using both small RNA blot analysis and locus-specific Real-Time PCR for the miR398 primary transcripts. We have validated the CSD1 and CSD2 genes as genuine targets in rice and tomato using RNA ligase-mediated 5' RACE assays. Our ability to confirm CCO as genuine target using modified 5'RACE assay in rice and tomato has not been successful. On the other hand, we have identified a copper chaperone for Cu/Zn superoxide dismutase (CCS) as a novel target for miR398 in rice and tomato. Co-expression of miR398 and CSD1/CSD2 genes in Nicotiana benthamiana leaves revealed that miR398 also regulate the expression of its target genes at the translational level, as has been reported for Arabidopsis miR398. Surprisingly miR398 levels in tomato and rice are not abundantly expressed as in Arabidopsis. Nevertheless, during diverse stress conditions that induce oxidative stress, miR398 levels were down-regulated, where as CSD1 and CSD2 levels were up-regulated. Nuclear-run on assays further confirmed that regulation of CSD1 and CSD2 transcripts were occurring at the post-transcriptional level and possibly dependent on miR398 levels. Transgenic rice and tomato plants overexpressing wild-type CSD1 and CSD2 and mCSD1 (miR-398 resistant CSD1) and mCSD2 (miR-398 resistant CSD2) genes have been generated. Molecular characterization of transgenic plants (T1) has been confirmed by using Q-PCR analysis and the selected lines with high abundance of transgene were grown for another generation. Subsequently T2 transgenic rice and tomato lines were analyzed for the abundance of the transgenes using Northern and western blot analysis. The analysis confirmed that the CSD1 or CSD2 gene expression is relatively higher in transgenic lines overexpressing miR398-resistant CSD1 or miR398-resistnat CSD2 genes, respectively, relative to wild-type transgenics. The T2 transgenic rice and tomato lines that had comparable levels of CSD1 or CSD2 expression and also activity as determined by enzyme assays, were selected and evaluated for their stress tolerance. The percent germination test under different salinity levels (0, 50, 100 and 150 mM NaCl), PEG stress, Cu2+ levels (0, 75, 125 and 175uM), methyl violgen (0 and 0.25 uM) suggested that the transgenic lines overexpressing miR398-resistant CSD1 or miR398-resistnat CSD2 genes showed improved tolerance compared to the transgenic lines that overexpress the wild-type CSD1 or CSD2 genes, which are susceptible to miR398-guided cleavage. Similar evaluations were also conducted in three-week-old seedlings of transgenic rice and tomato that are transferred to different stress conditions. The physiological assays (lipid peroxidation, ROS, net photosynthesis, chlorophyll and anthocyanin accumulation) of the seedlings also indicated the superior performance of miR398-resistant transgenic lines over the miR398-susceptible transgenics. PARTICIPANTS: Ramanjulu Sunkar (PI) Guosheng Li (Post-doc) Yong-fang Li (Post-doc) are members of the Department of Biochemitsry and molecular Biology at Oklahoma State University. Martha L. Orazco-Cardenas from University of California, Riverside, is also involved in this project. TARGET AUDIENCES: Scientists interested in miRNA biology and plant stress tolerance PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Identification of molecular mechanisms involved in abiotic stress tolerance is an important research topic in plant biology. Several genes have been shown to improve plant performance under stress conditions when overexpressed. Our strategy is based on the recent evidence that miR398 is a negative regulator of Cu/Zn superoxide dismutase genes (CSD1 and CSD2), which are considered as positive regulators of stress tolerance. This work formed as basis for investigating the miR398 and CSD genes in other crop plants. We have trained two post-doctoral fellows in probing miRNA-guided gene regulations including performing small RNA blot analysis, 5'-RACE assays, and molecular characterization of transgenic plants and physiological assays for determining stress tolerance.
Publications
- Sunkar, R., Jagadeeswaran, G. and Li, Y. (2011) Functions of microRNAs in plant stress responses. Trends in Plant Sciences, invited review (in review)
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Progress 08/01/09 to 07/31/10
Outputs
OUTPUTS: Transgenic rice and tomato plants overexpressing wild-type CSD1 and CSD2 and mCSD1 (miR-398 resistant CSD1) and mCSD2 (miR-398 resistant CSD2) genes have been generated. Molecular characterization of transgenic plants (T1) has been confirmed by using RT-PCR as well as Q-PCR and determined the level of superoxide dismutase gene expression in these transgenic plants. Transgenic rice or tomato plants that had substantially higher levels of SOD expression were selected and are currently being evaluated for stress tolerance. The percent germination is evaluated under different salinity levels (50, 100 and 150 mM NaCl) as well as under PEG stress. Similarly, germination studies also being conducted to analyze the response to oxidative stress conditions using methyl viologen or high levels of Cu2+. Seedling growth and other physiological parameters such as chlorophyll or anthocyanin levels under salt, oxidative stress as well as under cold stress are being carried out in transgenic tomato and rice. PARTICIPANTS: Ramanjulu Sunkar (PI) Guosheng Li (Post-doc) Martha L. Orazco-Cardenas (University of California, Riverside) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period. PARTICIPANTS: Ramanjulu Sunkar (PI), Guosheng Li (Post-doctoral fellow) and Martha L. Orazo-cardenas (university of California, Riverside) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We trained a new Post-doctoral fellow in probing microRNA-guided gene regulations including performing small RNA blot analysis, 5'-RACE assays, and molecular characterization of transgenic plants. We generated rice and tomato transgenic plants and determined the level of transgene expression. Currently, these transgenic plants are being analyzed for their stress tolerance under NaCl salinity, oxidative stress (high Cu2+ or methyl viologen) and cold stress conditions. We are testing both the germination response and early seedling development. Additionally, we recently identified that in addition to two Cu/Zn superoxide dismutases, miR398 is also targeting a chaperone for Cu/Zn superoxide dismutase in rice and we are characterizing the response of this gene under stress conditions.
Publications
- Ramanjulu Sunkar (2010). MicroRNAs with macro-effects on Plant stress responses. Seminars in Cell and Developmental Biology, 21, 805-811.
- Li YF., Zheng Y., Addo-Quaye, C. Li, Z., Saini, A., Jagadeeswaran G, Axtell, M. Zhang W, Sunkar R. (2010). Transcriptome-wide identification of microRNA targets in rice. Plant Journal 62, 742-759.
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Progress 08/01/08 to 07/31/09
Outputs
OUTPUTS: miR398 and it's target genes (CSD1 and CSD2) expression analysis has been completed both in tomato and rice using both small RNA or Northern blot analysis as well as Real-Time PCR. WE also have validated the CSD1 and CSD2 genes as genuine targets inr ice and tomato using RNA ligase-mediated 5' RACE assays. Surprisingly miR398 levels in tomato and rice are not expressed as abundantly as in Arabidopsis. Nevertheless during diverse stress conditions miR398 levels were down-regulated where as CSD1 and CSD2 levels were up-regulated. Nuclear-run on assays further confirmed that regulation of CSD transcripts were occurring at the post-transcriptional level and possibly dependent on miR398 levels. Tomato and rice transgneics expressing wild-type CSD1 and CSD2 and miR-398 resistant CSD1 and CSD2 genes have been successfully generated. Currently, these transgenic plants are being propagated to obtain T1 seeds. PARTICIPANTS: Ramanjulu Sunkar (PI) Yong-fang Li (Post-doc) Martha L. Orazco-Cardenas (University of California, Riverside) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Surprisingly miR398 levels in tomato and rice are not expressed as abundantly as in Arabidopsis. Nevertheless during diverse stress conditions miR398 levels were down-regulated where as CSD1 and CSD2 levels were up-regulated. Collectively, our results indicated that CSD1 and CSD2 genes are post-transcriptionally regulated and dependent on miR398 levels under stress conditions. The Post-doctoral fellow has been trained in probing microRNA-guided gene regulations in plants.
Publications
- 1. Guru Jagadeeswaran, Ajay Saini and Ramanjulu Sunkar (2008). Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis. Planta. 229:1009-14.
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Progress 08/01/07 to 07/31/08
Outputs
OUTPUTS: We have conducted the temporal expression analysis of microRNA398 and it's two targets genes (superoxide dsimutases, CSD1 and CSD2) in tomato. This has been also done in rice partly and the remaining experiments are in progress. We have trained a post-doctoral fellow in performing small RNA blot analysis and she optimized the epxression analysis in tomato as well as in rice. The Post-doctoral fellow also generated the constructs (wild-type CSD1 and CSD2 and mutated miR-398 resistant CSD1 and CSD2) necessary for transforming tomato and rice. PARTICIPANTS: Ramanjulu Sunkar (PI) Yong-fang Li (Post-doc) Martha L. Orazco-Cardenas (University of California, Riverside) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
we have accomplished the objective of determining the expression analysis of microRNA398 and superoxide dismutases genes in tomato and rice. The Post-doctoral fellow generated constructs (wild-type CSD1 and CSD2 and mutated miR-398 resistant CSD1 and CSD2) have been used for transforming tomato and rice. This helped us to generate tomato transgenics for all 4 constructs mentioned above. Currently we are performing molecular analysis on these transgenic plants to determine the level of trangene expression. Generation of transgenic rice plants is in progress.
Publications
- Shukla, L., Chinnusamy, V. and Ramanjulu Sunkar (2008). The Role of MicroRNAs and other Endogenous Small RNAs in Plant Stress Responses. Biochem. Biophys. Acta: Gene Regulatory Mechanisms, (in press).
- Ramanjulu Sunkar and Guru Jagadeeswaran, (2008). In silico identification of conserved miRNAs in large number of diverse plant species. BMC Plant Biology, 8:37.
- Ramanjulu Sunkar, Zhou, X., Zheng, Y., Zhang, W. and Zhu, J-K. (2008). Identification of novel and candidate miRNAs in rice by high throughput sequencing. BMC Plant Biology, 8:25.
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