Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan (Dimocarpus longan Lour.)

Abstract

Somatic embryogenesis (SE), which resembles zygotic embryogenesis, is an essential component of the process of plant cell differentiation and embryo development. Although microRNAs (miRNAs) are important regulators of many plant develop- mental processes, their roles in SE have not been thoroughly investigated. In this study, we used deep-sequencing, computational, and qPCR methods to identify, profile, and describe conserved and novel miRNAs involved in longan (Dimocarpus longan) SE. A total of 643 conserved and 29 novel miRNAs (including star strands) from more than 169 miRNA families were identified in longan embryogenic tissue using Solexa sequencing. By combining computational and degradome sequencing approaches, we were able to predict 2063 targets of 272 miRNAs and verify 862 targets of 181 miRNAs. Target annotation revealed that the putative targets were involved in a broad variety of biological processes, including plant metabolism, signal transduction, and stimulus response. Analysis of stage- and tissue-specific expressions of 20 conserved and 4 novel miRNAs indicated their possible roles in longan SE. These miRNAs were dlo-miR156 family members and dlo-miR166c* associated with early embryonic culture developmental stages; dlo-miR26, dlo-miR160a, and families dlo-miR159, dlo-miR390, and dlo-miR398b related to heart-shaped and torpedo- shaped embryo formation; dlo-miR4a, dlo-miR24, dlo-miR167a, dlo-miR168a*, dlo-miR397a, dlo-miR398b.1, dlo-miR398b.2, dlo-miR808 and dlo-miR5077 involved in cotyledonary embryonic development; and dlo-miR17 and dlo-miR2089*-1 that have regulatory roles during longan SE. In addition, dlo-miR167a, dlo-miR808, and dlo-miR5077 may be required for mature embryo formation. This study is the first reported investigation of longan SE involving large-scale cloning, characterization, and expression profiling of miRNAs and their targets. The reported results contribute to our knowledge of somatic embryo miRNAs and provide insights into miRNA biogenesis and expression in plant somatic embryo development.

Figure 1. Length distribution of unique sequences in longan.

Figure 2. Cleavage site mapping of miR398 target gene.

mRNA sequence of DlCSD2a is aligned with miR398. Nucleotides flanking the cleavage site are in bold. Numbers indicate the fraction of cloned PCR products terminating at different positions.

Figure 3. qPCR analysis of relative expressions of known and novel miRNAs, 5S rRNA, and U6 snRNA during longan SE.

The bar represents the scale of relative expression levels of miRNAs, and colors indicate relative signal intensities of miRNAs. Each column represents a sample, and each row represents a single miRNA. Samples: 1. friable-embryogenic callus(EC); 2. embryogenic callus II(EC II); 3. incomplete compact pro-embryogenic cultures(ICpEC); 4. compact pro-embryogenic cultures(CpEC); 5. globular embryos(GE); 6. heart-shaped embryos(HE); 7. torpedo- shaped embryos(TE); 8. cotyledonary embryos(CE); 9. mature embryos(ME).

Figure 4. Morphology of embryogenic calli and embryos during the six sequential developmental stages of longan SE.

Developmental stages are indicated at the left of each row. The bars in each phenotypic class are indicated at the middle of each image. The morphology of embryogenic cultures (EC-b, ICpEC, GE, HE, TE, CE-a) were observed using an inverted Leica DMIL LED microscope, except for TE(bar = 500 µm), the bars of others are 200 µm; the images of EC-a and CE-b were both obtained under an Leica DFC295, bars, 600 µm; EC, ICpEC, GE, HE, and TE were cultured on MS medium supplemented with 1 mg/L, 0.5 mg/L, 0.1 mg/L, 0.06 mg/L and 0.03 mg/L 2,4-D, respectively; and the CE was cultured on MS medium.