Cryo-Treatment Enhances the Embryogenicity of Mature Somatic Embryos via the lncRNA-miRNA-mRNA Network in White Spruce

Abstract

In conifers, somatic embryogenesis is uniquely initiated from immature embryos in a narrow time window, which is considerably hindered by the difficulty to induce embryogenic tissue (ET) from other tissues, including mature somatic embryos. In this study, the embryogenic ability of newly induced ET and DNA methylation levels was detected, and whole-transcriptome sequencing analyses were carried out. The results showed that ultra-low temperature treatment significantly enhanced ET induction from mature somatic embryos, with the induction rate from 0.4% to 15.5%, but the underlying mechanisms remain unclear. The newly induced ET showed higher capability in generating mature embryos than the original ET. DNA methylation levels fluctuated during the ET induction process. Here, WGCNA analysis revealed that OPT4, TIP1-1, Chi I, GASA5, GST, LAX3, WRKY7, MYBS3, LRR-RLK, PBL7, and WIN1 genes are involved in stress response and auxin signal transduction. Through co-expression analysis, lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might bind to pre-novel_miR_339 to promote the expression of WRKY7 genes for stress response; LAX3 could be protected by lncRNAs MSTRG.1070680.1 and MSTRG.33602.1 via serving as sponges for novel_miR_495 to initiate auxin signal transduction; lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might serve as sponges for novel_miR_527 to enhance the expression of Chi I for early somatic embryo development. This study provides new insight into the area of stress-enhanced early somatic embryogenesis in conifers, which is also attributable to practical applications.

Keywords: conifer; cryo-treated mature somatic embryos; liquid nitrogen; lncRNAs; miRNAs; whole transcriptome.

Figure 1

Induction process of cryo-treated mature somatic embryos (CRSEs) and normal mature somatic embryos cultured at room temperature (RTSEs): (a,c,e,g,i) show tissues sampled on day 0, 1, 10, 15, and 20 with explants of CRSE; (b,d,f,h,j) show tissues on day 0, 1, 10, 15, and 20 of RTSE. All bars = 500 μm.

Figure 2

Microscopic observation of the change in cryo-treated mature somatic embryos (CRSEs) and normal mature somatic embryos cultured at room temperature (RTSEs) during callus induction: (a,c,e,g,i) show tissues sampled on day 0, 1, 5, 10, and 13 of CRSE; (b,d,f,h,j) show tissues sampled on day 0, 1, 5, 10, and 13 of RTSE. All bars = 50 μm.

Figure 3

Induction rate, embryonic ability, DNA methylation level, and DNMTs expression heatmap: (a) average induction rate of CRSE and RTSE; (b) the total number of somatic embryos per gram of ET and NET; (c) global DNA methylation level of samples on day 0, 1, 5, 10, and 20 of CRSE (CR) and RTSE (RT); (d) heatmap of DNA methyltransferases genes chromatinmethylase (CMT), domains rearranged methylase 2 (DRM2), and methyltransferase (MET). Asterisk and different letters represented significance (p < 0.05).

Figure 4

Analysis of differentially expressed mRNAs (DEGs): (a) sample cluster of 6 samples with three replicates, including CR0D, CR1D, CR10D, RT0D, RT1D, and RT10D; (b) cluster of DEGs in CR0D, CR1D, CR10D, RT0D, RT1D, and RT10D.

Figure 5

KEGG analysis of differentially expressed mRNAs (DEGs).

Figure 6

WGCNA analysis of DEGs: (a) cluster dendrogram of WGCNA analysis. Branches with different colors correspond to 10 different modules; (b) expression profile of DEGs in 6 samples of blue module; (c) network construction of DEGs in blue module, and 11 hub genes were selected in red font.

Figure 7

qPCR verification of mRNAs. qPCR results of WRKY transcription factor 7 (WRKY7), wound-induced protein WIN1 (WIN1), auxin transporter-like protein 3 (LAX3), Myb-related protein S3 (MYBS3), gibberellin-regulated protein 5 (GASA5), and glutathione S-transferase (GST). RT represents RTSE, and CR represents CRSE. The columns represent the RNA-seq expression level related to the left axis, while the lines represent the qPCR expression level correlated with the right axis.

Figure 8

Co-expression analysis of lncRNAs and mRNAs in blue module of WGCNA results: (A) Venn analysis of up- and downregulated DElncRNAs of CR0D vs. CR1D, CR0D vs. CR10D, RT0D vs. RT1D, and RT0D vs. RT10D; (B) cluster analysis of the common lncRNAs in Venn analysis; (C) Co-expression network of lncRNAs and mRNAs in blue module; the color of circle and line represent coefficient and p-value, respectively; (D) Co-expression network of lncRNAs and hub genes; the color of diamond represents coefficient.

Figure 9

qPCR verification of lncRNAs and miRNAs: (a) qPCR verification of 3 lncRNAs, including MSTRG.33602.1, MSTRG.505746.1, and MSTRG.1070680.1; (b) qPCR verification of 3 miRNAs, including novel_miR_339, novel_miR_495, and novel_miR_527. RT represents RTSE, and CR represents CRSE. The columns represent the RNA-seq expression level related to the left axis, while the lines represent the qPCR expression level correlated with the right axi.

Figure 10

Analysis of DEmiRNAs (differentially expressed microRNAs) and targeted genes in blue module of WGCNA results: (a) hierarchical clustering of DEmiRNAs analysis; (b) network of miRNAs and targeted genes of blue module; blue triangle represents miRNAs, while yellow square represents targeted genes; (c) network of miRNAs and targeted hub genes; blue triangle represents miRNAs, and yellow square represents targeted genes.

Figure 11

The proposed mechanism in the induction process of CRSE. The proposed mechanism in the induction process of CRSE, including oligopeptide transporter 4 (OPT4), aquaporin TIP1-1 (TIP1-1), class I chitinase (Chi I), gibberellin-regulated protein 5 (GASA5), glutathione S-transferase (GST), auxin transporter-like protein 3 (LAX3), LRR receptor-like serine/threonine-protein kinase At1g07650 (LRR-RLK), serine/threonine-protein kinase PBL7 (PBL7) and x wound-induced protein WIN1 (PBL7), WRKY transcription factor 7 (WRKY7), Myb-related protein S3 (MYBS3), MSTRG.505746.1, MSTRG.1070680.1, MSTRG.33602.1, pab-miR391c, pab-miR3627d, novel_miR_339, novel_miR_47, and novel_miR_495.