FUNCTIONAL ANALYSES OF ARABIDOPSIS RIBONUCLEOTIDE REDUCTASE SMALL SUBUNIT GENE FAMILY
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A fundamental question in plant development is how cell division events are coordinately regulated in the context of growth and development. To address this question, I chose to study a pleiotropic mutant, tso2, which exhibited developmental defects including callus-like floral organs and fasciated shoot meristem. I isolated the TSO2 gene and showed it encodes the small subunit of ribonucleotide reductase (RNR). RNR catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA synthesis. Subsequently, I showed that tso2 mutants reduce the dNTP levels.
To understand why tso2 mutants, defective in this essential process, are still viable, I identified two homologs of TSO2, R2A and R2B in Arabidopsis. Mutations in R2A and R2B were isolated using a reverse genetic approach. While r2a, r2b single mutants or r2a r2b double mutants fail to display any visible phenotype, r2a and r2b mutations can enhance tso2 genetically, resulting in seedling lethality and embryonic lethality in tso2 r2a and tso2 r2b, respectively. Overexpression of either R2A or R2B can rescue the tso2 mutants, suggesting that the three R2 genes are functionally redundant.
In addition to the developmental defects, tso2 mutants were more sensitive to HU (hydroxyurea) and UV-C, indicating that TSO2 plays a major role in DNA repair. In tso2 r2a double mutant seedlings, increased DNA damage accumulates, leading to massive programmed cell death. In addition, release of transcriptional gene silencing was observed in tso2 r2a double mutants, suggesting that DNA damage can lead to epigenetic instability. To further identify regulators of RNR and novel components of plant DNA damage response pathways, 18 independent tso2 suppressors were isolated in a genetic screen. These suppressors fall into at least four different complementation groups.
My genetic and molecular characterization of TSO2 is the first functional study of RNR in plants. My results indicated that plants could initiate programmed cell death in response to DNA damage. The developmental defects in tso2 mutants are caused by epigenetic instability and aberrant cell division. The isolation of potential tso2 suppressors will be crucial to the understanding of plant DNA damage response pathway, an understudied area in plant biology.