IC4R005-lncRNA-2016-19769571

Project Title

• Novel drought-responsive regulatory coding and non-coding transcripts from Oryza Sativa L.

The Background of This Project

• Drought-stress can cause major economic loss and is a serious issue to address in agriculture. Defining the molecular pathways in how a plant responds to droughtstress may prove valuable in developing new drought-resistant plants. In this study, the researchers identified several novel drought-responsive regulatory coding and noncoding transcripts in rice, Oryza sativa L., using the next generation sequencing (NGS) technique and bioinformatics analyses.

Plant Culture & Treatment

• Rice plants, O.sativa L. Nipponbare, were acquired from the Rural Development Administration of Korea (RDA). The rice plants were cultured in Yoshida solution at pH 5.8 and maintained in a temperaturecontrolled culture room at 29 ℃ under 16 h/8 h light/dark conditions. Rice plants at the three-leaf stage were subjected to drought-stress for 1 and 6 h by removal of the culture solution. Untreated plants were used as control. After treatment, entire plants were immediately transferred into liquid nitrogen.

Research Findings

• A total of 1080 and 4017 DEGs were found at 0–1 h and 0–6 h data points, respectively. Histograms and volcano plots of expression fold changes are shown in Fig. 1. The DEG analysis showed that the number of DEG increased as drought-stress continued. This observation showed that when the plant was exposed to a longer period of drought-stress, transcriptional changes were bigger for the experiment.

Figure 1. a For 0-to-1 h time duration, the number of DEGs vs. gene expression log2 fold change (left figure) and volcano plot (right figure). Each dot in the volcano plot represents a gene by its fold change and its significance level by -log10(p-value) b For 0–1 h time duration, the number of DEGs vs. Gene expression log2 fold change (left figure) and volcano plot (right figure)

• Among the 4017 DEGs from the 0-to-6 h data set, 1472 genes were activated and 2545 genes were suppressed. We investigated the known functions of the activated/suppressed differentially expressed genes under drought-stress by Gene Ontology (GO) enrichment analysis. The result is shown in Table 1.

Table 1. Enriched functions of activated and suppressed DEGs under drought-stress

• After filtering out TFs previously reported in drought-stress studies, we selected 68 TFs as candidates with unknown functions for further study. We performed qRT-PCR on the 68 TFs and validated 18 of them (Table 2). The expression level of all 18 coding TFs was increased after 1 and 6 h drought treatment compared to 0 h control.

Table 2. Novel/function-unknown drought-stress responsive coding transcripts.

• The researchers selected 10 novel lncRNAs for validation via qRT-PCR (Table 3).

Table 3. Drought-responsive novel long noncoding RNAs. Fold change is the ratio of expression level at 0–6 h

• As shown in Fig. 2, the novel miRNA and an existing miRNA, osa-miR169 g, are arranged in phase in a hairpin sequence. This result is consistent with the previous research in Arabidopsis where multiple small RNAs originated and are arranged in phase within a hairpin sequence. The expression level of os-miR9898 was down-regulated by 5.5-fold at 6 h compared to 0 h (Fig. 3). As a result, we selected one novel miRNA for validation via qRT-PCR (Fig. 2).

Figure 2. Hairpin structure of novel un-annotated droughtresponsive miRNA hairpin structure. Color represents the folding intensity between match pairs

Figure 3. Novel miRNA transcript qRT-PCR.

Labs working on this Project

• Department of Biomedical Sciences, Sunmoon University, Asan 336-708, Korea
• Department of Computer Science and Engineering, Seoul National University, Gwanak-Gu, Seoul 151-744, Republic of Korea
• Interdisciplinary Program in Bioinformatics, Seoul National University, Gwanak-Gu, Seoul 151-747, Republic of Korea
• Bioinformatics Institute, Seoul National University, GwanakGu, Seoul 151-747, Republic of Korea

Corresponding Author

• Hawk-Bin Kwon: hbkwon@sunmoon.ac.kr