Transcriptome Profiling Reveals Role of MicroRNAs and Their Targeted Genes during Adventitious Root Formation in Dark-Pretreated Micro-Shoot Cuttings of Tetraploid Robinia pseudoacacia L

Abstract

Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L^-1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1-1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants.

Keywords: RNA-seq; adventitious rooting; dark pretreatment; miRNA-seq; tetraploid Robinia pseudoacacia L..

Figure 1

Histological observations of dark-pretreated micro-shoot cuttings (IBA and non-IBA) of tetraploid R. pseudoacacia L. during AR formation process at 0, 12, 36, 48, and 72 HAE, in which only treated micro-shoots were subjected to 0.2 mg L⁻¹ IBA. (A) Histological observations of control (non-IBA), dark-pretreated micro-shoot cuttings, with no changes at mentioned HAE. (B) Histological observations of treated (IBA), dark-pretreated micro-shoot cuttings with apparent changes at HAE. Yellow arrows indicate the cambium layer, red arrows represent the first mitotic cell divisions and first AR primordium development on the cambium layer.

Figure 2

The number of differentially expressed (DE) microRNAs in dark-pretreated micro-shoot cuttings of tetraploid R. pseudoacacia L. during IBA-dependent AR formation in seven comparison group libraries (HAE0-vs-HAE12, HAE0-vs-HAE36, HAE0-vs-HAE48, HAE0-vs-HAE72, HAE12-vs-HAE36, HAE36-vs-HAE48, and HAE48-vs-HAE72). (a) Total upregulated and downregulated DE miRNAs expressed in seven comparison group libraries. (b) Venn diagrams showing the number of the differentially expressed miRNAs in the seven comparison group libraries. (c) Heatmap showing the fold changes of miRNAs in comparison group libraries.

Figure 3

Expression profile and identification of DE miRNAs during IBA-dependent AR formation in dark-pretreated micro-shoot cuttings of tetraploid R. pseudoacacia L. The figure depicts the shared and unique (known and novel) DE miRNAs of the associated transcriptome analyses. The overlap between the DE miRNAs was identified following 0 HAE, 12 HAE, 36 HAE, 48 HAE, and 72 HAE from the donor plant. The y-axis shows the number (0–90) of miRNA interactions. The x-axis represents the total number of miRNAs expressed in the first four comparison group libraries. Black circles show the common DE miRNAs in different comparison groups.

Figure 4

Hierarchical clustering of differentially expressed miRNAs in dark-pretreated tetraploid R. pseudoacacia L. during IBA-dependent AR formation. (a) Heatmap showing the number of the known differentially expressed upregulated and downregulated miRNAs in the four comparison groups. (b) Heatmap showing the expression pattern of the novel differentially upregulated and downregulated miRNAs. (c) A volcano plot showing the DE miRNAs in the HAE0-vs-HAE72 comparison group libraries. The clusters were generated based on the Pearson correlation coefficient of normalized miRNA expression values. Bars indicate log2 (TPM + 1) values.

Figure 5

KEGG classification of differentially expressed genes (DEGs) and differentially expressed microRNAs in HAE0-vs-HAE12 and HAE0-vs-HAE72 comparison libraries. (a) The number of DEGs in HAE0-vs-HAE12 libraries. (b) The number of DE miRNAs in HAE0-vs-HAE72 libraries. The x-axis represents the number of DEGs and DE miRNAs. The y-axis shows the KEGG pathway terms in DEGs and DE miRNAs.

Figure 6

Co-expression network of the miRNAs and their targeted genes expressed in dark-pretreated micro-shoot cuttings of tetraploid R. pseudoacacia L. during IBA-dependent AR formation. Co-expression network of miRNAs and their targets during plant hormone signal transduction and sucrose metabolism pathways at five time points.

Figure 7

Expression analysis of ten miRNAs and their twelve target genes obtained from dark-pretreated micro-shoot cuttings of tetraploid R. pseudoacacia L. at 0, 12, 36, 48, and 72 HAE. (a–f) The genes and their respective miRNA expression during the auxin signaling pathways. (g–l) The genes and their respective miRNA expression during the starch and sucrose metabolism pathway. The data presented are an average of three technical replicates.