Transcriptomic analysis of OsRUS1 overexpression rice lines with rapid and dynamic leaf rolling morphology

Abstract

Moderate leaf rolling helps to form the ideotype of rice. In this study, six independent OsRUS1-GFP overexpression (OsRUS1-OX) transgenic rice lines with rapid and dynamic leaf rolling phenotype in response to sunlight were constructed. However, the mechanism is unknown. Here, RNA-Seq approach was utilized to identify differentially expressed genes between flag leaves of OsRUS1-OX and wildtype under sunlight. 2920 genes were differentially expressed between OsRUS1-OX and WT, of which 1660 upregulated and 1260 downregulated. Six of the 16 genes in GO: 0009415 (response to water stimulus) were significantly upregulated in OsRUS1-OX. The differentially expressed genes between WT and OsRUS1-OX were assigned to 110 KEGG pathways. 42 of the 222 genes in KEGG pathway dosa04075 (Plant hormone signal transduction) were differentially expressed between WT and OsRUS1-OX. The identified genes in GO:0009415 and KEGG pathway dosa04075 were good candidates to explain the leaf rolling phenotype of OsRUS1-OX. The expression patterns of the 15 genes identified by RNA-Seq were verified by qRT-PCR. Based on transcriptomic and qRT-PCR analysis, a mechanism for the leaf rolling phenotype of OsRUS1-OX was proposed. The differential expression profiles between WT and OsRUS1-OX established by this study provide important insights into the molecular mechanism behind the leaf rolling phenotype of OsRUS1-OX.

Figure 1

Leaf rolling morphology of WT and OsRUS1-OX in response to sunlight. (A) Morphology of the WT and OsRUS1-OX under sunlight, scale bar 2 cm; (B) The rapid leaf expanding in OsRUS1-OX when the sunlight was shaded, scale bar 2 mm; (C) The rapid leaf rolling in OsRUS1-OX in response to sunlight, scale bar 2 mm. WT, the wildtype of ZhongHua11; OsRUS1-OX, the OsRUS1-GFP Overexpression transgenic line.

Figure 2

Pearson correlation between WT and OsRUS1-OX RNA-Seq data.The change of the square (R²) value of the correlation coefficient of Pearson is indicated by the change of the blue color. The deeper color indicates a bigger R² value and a higher correlation between samples. WT, the wildtype of ZhongHua11; OsRUS1-OX, the OsRUS1-GFP Overexpression transgenic plant.

Figure 3

Hierarchical clustering of the differentially expressed genes between WT and OsRUS1-OX. The RNA-seq data derived from WT and OsRUS1-OX based on log₁₀(FPKM + 1) values were utilized. The blue bands represent down regulated genes, and the red bands represent up regulated genes. WT, the wildtype of ZhongHua11; OsRUS1-OX, the OsRUS1-GFP Overexpression line.

Figure 4

The k-means clustering of differentially expressed genes between WT and OsRUS1-OX. The eight major clusters obtained by K-means algorithm, representing upregulated (1, 3, 5), and downregulated (2, 4, 6, 7, 8) clusters. Expression ratios are expressed as Log₂. WT, the wildtype of ZhongHua11; OsRUS1-OX, the OsRUS1-GFP Overexpression transgenic line.

Figure 5

Histogram of Gene Ontology (GO) classification of differential expressed genes between WT and OsRUS1-OX. The results are summarized in three main categories: biological process (BP), cellular component (CC), and molecular function (MF), shown in green, red, and blue, respectively. The Y-axis indicates enriched Gene Ontology (GO) terms; the X-axis indicates the number of differential genes in a category; *, significantly enriched GO term. A. significantly enriched Gene Ontology (GO) terms in the upregulated genes in the leaves of OsRUS1-OX as compared to WT; B. significantly enriched Gene Ontology (GO) terms in the downregulated genes in the leaves of OsRUS1-OX as compared to WT OsRUS1-OX. WT, the wildtype of ZhongHua11; OsRUS1-OX, the OsRUS1-GFP Overexpression transgenic line.

Figure 6

Mapman analysis of differentially expressed genes of WT vs OsRUS1-OX. (A) metabolism overview; (B) regulation overview; (C) transport overview; (D) cellular response overview; (E) photosynthesis. Up- and Down- regulated DEGs are represented with blue and red squares, respectively with log₂ (Fold Change values).

Figure 7

Verification of differentially expressed genes between WT and OsRUS1-OX by qRT-PCR. Note: Histograms represent the results of the qRT-PCR assays, using the 2^−ΔCt algorithm, with the scale on the left ordinate of each graph. Red dots represent the results of the FPKM analyses, with the scale on the right ordinate of each graph. WT, the wildtype of ZhongHua11; OsRUS1-OX, the OsRUS1-GFP Overexpression transgenic plant.

Figure 8

A working model explaining the rapid and dynamic leaf rolling in the OsRUS1-OX in response to sunlight. Note: The expressions of some genes in mesophyll cells (green cycle) are regulated due to the overexpression of OsRUS1-GFP. The transgenic plants become more sensitive to sunlight. The sunlight will trigger the pathway to cause the leaf rolling of OsRUS1-OX. The WT is not as sensitive to sunlight as OsRUS1-OX, and the pathway will not be triggered under the same conditions. If the sunlight is shaded, although the pathway is still there, it is not activated, and the bulliform cells of OsRUS1-OX will be refilled with water to cause leaf expansion.