Cloning And Functional Analysis Of OsDARE1 Controlling Adventitious Root Development In Rice

Adventitious roots are main components of fibrous root system and important for crops including rice to anchor and absorb water and nutrition. However, to date the regulatory mechanism of adventitious root development is poorly understood. In this study, we isolated a mutant Osdarel which was defective in adventitious root emergence from an EMS mutant library of Kasalath. The pheonotypic characterization of the mutant, cloning of the gene and its function analysis were conducted. The results are summerized as follows:1. The initiation and development of adventitious root primordia in the mutant is normal. Similar number of adventitious root primordia was observed in the mutant compared to the wild type (WT). Further analysis revealed that adventitious root primordia could not emerge from the epidermis in the mutant. Therefore, the mutant was named as Osdarel (defective in adventitious root emergence 1).2. The OsDAREl locus was mapped on short arm of chromosome 2 through map-based cloning. The gene (LOC_Os02g07120) encodes an ortholog of Arabidopsis CANDl (cullin-associated and neddylation-disassociated 1). The complementation analysis further approved that the mutation was caused by a point mutation at 533bp of ORF of OsDAREl.3. The OsDAREl is a nuclear protein. The OsDAREl expresses in various organs, including root, stem-base, stem, leaf and flowers. The fused gene of OsDAREl:.OsDAREl::GUS was transformed into the mutant and sufficient to rescue Osdarel mutant phenotype. It was found that the expression of OsDAREl was spatial and temporal. In the stem base, GUS staining was not detected until adventitious root architecture established where it was detected only in the root cap of adventitious root.4. The LCM (Laser Captured Microdissection) analysis showed that before establishment of fundamental organization of adventitious root primordia, the expression of cell cycle genes were not significantly different between WT and Osdarel. However, after the establishment, distinct expression difference of cell cycle gene for G2/M transition was detected in Osdarel, with activation of the negative regulator KRP2 and repression of the positive regulator CDKB;2. While the expression of regulators for Gl/M transition and S phase (such as RBI and CDKD;1) were not changed compared to WT. This indicates that the failure of formation of mature adventitious root in Osdarel is due to the ceasession of G2/M transition of cell cycle in the meristem.5. GUS report gene driven by DR5 promoter is responsible for auxin transcriptional signaling in plant. DR5::GUS in adventitious root primordia of Osdarel was normal during the formation of primordia, and then rapidly disappeared in the central cylinder of adventitious root leaving smear expression pattern in the root cap. This indicates that the auxin signaling was altered after root architecture established in Osdarel.6. Adventitious root in Osdarel can be partially rescued by exogenous auxin. This suggests that OsDAREl may modulate auxin signaling to regulate G2/M transition of cell cycle in adventitious root meristem, and consequently to affect adventitious root emergence.