Knock-down of a RING finger gene confers cold tolerance

Abstract

The plant-specific RING-domain finger proteins play important roles in plant development and stress responses. We recently identified and functionally characterized a stress-induced gene OsSRFP1 (Oryza sativa Stress-related RING Finger Protein 1) from rice. We showed evidences of the biotechnological potential of the suppression of OsSRFP1 expression in conferring cold tolerance. The increased cold tolerance of OsSRFP1 knock-down plants was associated with higher amounts of free proline and activities of antioxidant enzymes. In vitro ubiquitination assays showed that OsSRFP1 possessed E3 ubiquitin ligase activity. Some predicted interacting partners of OsSRFP1 might be the substrates for OsSRFP1-mediated protein degradation. Interestingly, OsSRFP1 had trans-activation activity, suggesting the dual roles of OsSRFP1 in post-translational and transcriptional regulations in stress responses.

Keywords: E3 ubiquitin ligase; cold stress; rice; transcription factor.

Figure 1.

Silencing of OsSRFP1 confers cold tolerance in rice. Phenotypes of 2-week-old transgenic rice plants and the wild type before and after cold treatment, followed by recovery for 2 weeks.

Figure 2.

Distribution of stress-related cis-acting elements in the 2,000 bp promoter region of OsSRFP1. The 2,000-bp upstream sequence of OsSRFP1 was analyzed by Matinspector program at Genomatix website (http://www.genomatix.de/). Different cis-elements for TFs were labeled with different colors.

Figure 3.

Prediction of OsSRFP1 interacting proteins. OsSRFP1 protein sequence was analyzed with STRING 9.0 at (http://string-db.org). The results showed the presence of a number of proteins interacting with OsSRFP1, which are presented in the table below the figure.

Figure 4.

In Silico expression analysis of predicted OsSRFP1-interacting proteins under stress treatment in rice. Gene ID of predicted OsSRFP1 interacting proteins were analyzed on Affymetrix microarray datasets (http:/ricearray.org). (A) Heatmap of expression analysis of predicted OsSRFP1 interacting proteins under stress treatment. Heatmap scale bar: 5, 9, 13. (B) Expression graph analysis of predicted OsSRFP1 interacting proteins under stress treatment. Array element ID: Os.49023.1.S1_x_at is representative of OsSRFP1, Os.10901.1.S1_a_at is representative of the gene coding the MYB family transcription factor, Os.53502.1.S1_at is representative of the gene coding the CHCH domain containing protein. The data in the graph indicate the log2 fold of the microarray data in rice seedlings under drought, salt, and cold stress relative to that under control condition. Data represent means and standard errors of 3 replicates.

Figure 5.

Growth of OsSRFP1-RNAi transgenic rice. (A) qRT-PCR identification of OsSRFP1-RNAi transgenic rice lines. Data represent means and standard errors of 3 replicates. (B) The growth of OsSRFP1-RNAi transgenic rice lines at 6-leaf stage. Scale bars = 10cm.