Gradual increase of miR156 regulates temporal expression changes of numerous genes during leaf development in rice

Abstract

The highly conserved plant microRNA, miR156, is an essential regulator for plant development. In Arabidopsis (Arabidopsis thaliana), miR156 modulates phase changing through its temporal expression in the shoot. In contrast to the gradual decrease over time in the shoot (or whole plant), we found that the miR156 level in rice (Oryza sativa) gradually increased from young leaf to old leaf after the juvenile stage. However, the miR156-targeted rice SQUAMOSA-promoter binding-like (SPL) transcription factors were either dominantly expressed in young leaves or not changed over the time of leaf growth. A comparison of the transcriptomes of early-emerged old leaves and later-emerged young leaves from wild-type and miR156 overexpression (miR156-OE) rice lines found that expression levels of 3,008 genes were affected in miR156-OE leaves. Analysis of temporal expression changes of these genes suggested that miR156 regulates gene expression in a leaf age-dependent manner, and miR156-OE attenuated the temporal changes of 2,660 genes. Interestingly, seven conserved plant microRNAs also showed temporal changes from young to old leaves, and miR156-OE also attenuated the temporal changes of six microRNAs. Consistent with global gene expression changes, miR156-OE plants resulted in dramatic changes including precocious leaf maturation and rapid leaf/tiller initiation. Our results indicate that another gradient of miR156 is present over time, a gradual increase during leaf growth, in addition to the gradual decrease during shoot growth. Gradually increased miR156 expression in the leaf might be essential for regulating the temporal expression of genes involved in leaf development.

Figure 1.

Expression of miR156 is gradually increased during leaf development. A to C, Expression levels of miR156 in sequentially developed leaves at three different stages: seedling (25 DAG), tillering (40 DAG), and graining (100 DAG). As shown in the photographs of rice plants on the right, the leaf samples were numbered according to the order of emergence from the tiller. The relative expression levels are indicated at the bottom of the blots except for weak bands. The labeled leaves were initiated at the adult vegetative phase, except that L3/L4 were intermediate juvenile/adult leaves of the main tiller. D, Comparison of the maximal miR156 level in leaves at different shoot stages. [See online article for color version of this figure.]

Figure 2.

miR156 level is positively correlated with leaf developmental time. A, Expression of miR156 during leaf development. Rice leaves L4, L6, L8, L9, L10, and flag leaf from the main tiller were collected every 5 d after their emergence. B, Relative expression of miR156 appears to be correlated to leaf developmental time in the flag leaf. C, Comparison of the maximal level of miR156 in leaves (L4 and L5) and 3-d-old shoot.

Figure 3.

Expression of miR156 in Md/Mh and wild-type leaves. Wild-type leaves are labeled WT1 to WT3. The RNA samples labeled with asterisks were also used for microarray hybridization. Relative levels of miR156 are shown on the bottom of blots except for weak bands.

Figure 4.

Temporal expression of miR156-targeted OsSPLs during leaf development in Md and wild-type (WT) plants. Error bars indicate sd (n = 3).

Figure 5.

Summary of miR156-regulated genes in different leaves. A, Venn diagram representation of miR156 up- and down-regulated genes in L6 (later-emerged young leaf) to L3 (early-emerged old leaf). B, Most miR156-regulated genes have temporal expression in the wild type (WT). Time positive indicates that gene expression is increased from L6 to L3 in the wild type; time negative indicates that gene expression is decreased from L6 to L3 in the wild type. C, Distribution of logFC of miR156-regulated genes in the wild type and Md. LogFC indicates the fold change of each gene from young to old leaf in these plants. D, Heat map shows logFC of different plants. Color indicates the value of logFC as shown on the bar at bottom. E, Clusters of miR156-regulated gene expression patterns.

Figure 6

Confirmation of the results of miR156-regulated genes from microarray analysis by RT-qPCR. Error bars indicate 95% confidence interval. WT, Wild type.

Figure 7.

Expression of miR156-regulated microRNAs temporally during leaf development. A, Relative levels of 15 microRNAs in different leaves were monitored by stem-loop RT-qPCR. RNAs are from the same samples as in Figure 2. Error bars indicate sd (n = 3). WT, Wild type. B, Cluster analysis of microRNAs according to their temporal expression in the wild type. The relative expression level is indicated by the color scale. C, The stem-loop RT-qPCR results were confirmed by small RNA gel blotting of miR164.

Figure 8.

miR156-OE changed leaf development. A to D, Dynamic changes of plant height, leaf number, tiller number, and leaf initiation rate of wild-type (WT)/NC and Md/Mh plants (n = 12). Error bars indicate se. E to J, Comparison of Md/Mh and wild-type/NC plants in leaf shape (length and width) at three representative stages of rice (n = 6). Error bars indicate sd. K, Md/Mh and wild-type/NC plants at the flowering stage (90 DAG). [See online article for color version of this figure.]