Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment

Abstract

Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H2 treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA) libraries to identify global transcriptome changes at different time points during H2 treatment of larch pro-embryogenic masses (PEMs). A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS) homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H2 during somatic embryogenesis.

Molecular Dissection of the Regenerative Capacity of Forest Tree Species: Special Focus on Conifers

“…This effect of the hydrogen treatment was linked to the expression of stress-related genes in larch PEMs, reduced reactive oxygen species (ROS) levels, and enhanced antioxidant enzyme activity. These findings indicated that increased rates of active PEMs and normal somatic embryos may be, at least partially, a result of reduced ROS levels after hydrogen treatment (Liu et al, 2016). Up-regulation of flavonoid biosynthesis genes and related transcription factors in the EMs of Norway spruce may have promoted embryo development by regulating polar auxin transport and the functions of housekeeping enzymes, in addition to the more general role of flavonoids in ROS scavenging and abiotic stress resistance (Reza et al, 2018).…”

“…However, they still failed to develop into mature somatic embryos, indicating that additional factors or developmental pathways are involved in embryo maturation (Park et al, 2010). The ability to control oxidative stress to prevent oxidative damage and maintain cell and protein structure appeared to be a key component for embryogenesis induction in larch (Liu et al, 2016). Treatments with hydrogen, an antioxidant molecule, increased the proportion of active pro-embryogenic cells in PEMs, and induced normal somatic embryos.…”

“…In Chinese fir, SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)1-2 and WOX13 genes, the former encoding a LRR receptor-like kinase involved in the induction of SE competence in culture (Schmidt et al, 1997), were predominantly expressed in PEMs, the transition to late-stage PEMs associated with stress-related responses, and pro-embryo transition (Zhou et al, 2017). The enhanced performance of larch PEMs exposed to hydrogen was associated with antioxidant defense signaling, and thus, with the regulation of redox homeostasis (Liu et al, 2016). Compared with controls, the hydrogen-treated larch PEMs also showed significant differential expression of BBM and WOX , which encode critical regulators of SE, and genes encoding endonucleases, exonucleases, cysteine proteases, aspartic proteases, and metacaspases, which are involved in programmed cell death (PCD).…”

Transcriptional and post-transcriptional regulation of the miR171-LaSCL6 module during somatic embryogenesis in Larix kaempferi

“…The genomic DNA of L. kaempferi was isolated from the embryonal-suspensor mass with the CTAB plant genome DNA rapid extraction kit (Aidlab, China) according to the manufacturer's protocols. MIR171 sequences were amplified from genomic DNA with Platinum® Taq DNA polymerase (Invitrogen, USA) with specific primers (Table S1), which were designed based on the annotation of miRNA (Liu et al 2016;Zhang et al 2012) of L. kaempferi. The PCR products were purified with the Gel Extraction kit (Tiangen, China), ligated into the pEASY®-T1 simple cloning vector (Transgen, China), and sequenced.…”

Research Tools for the Functional Genomics of Plant miRNAs During Zygotic and Somatic Embryogenesis

“…miRNA profiling using NGS has revolutionised miRNA analysis and there are still new instruments, sequencing platforms, and new methods that appear, such as sRNA-seq using single-cell sequencing [ 81 ] which provide new opportunities for analyses also during embryogenesis. Until now, sRNA-seq analyses have been performed during embryogenesis in Picea sprus [ 82 ], P. pinaster [ 39 ], Picea glauca [ 30 ], Triticum aestivum [ 83 ], Arabidopsis [ 28 , 30 , 35 , 38 , 41 , 84 ], Dimocarpus longan [ 85 ], Zea mays [ 86 ], Citrus sinensis [ 87 ], Larix leptolepis [ 88 ], Gossypium hirsutum [ 89 , 90 ], Phyllostachys heterocycla [ 91 ], Oryza sativa [ 92 ], and Lilium pumilum [ 93 ] (for SE reviewed in [ 94 ]). Plant embryogenesis is challenging to analyse due to the small, early embryos that are deeply embedded in the maternal tissues, which often results in RNA contamination from maternal tissue, and due to difficult distinguishing of the cells that are undergoing SE within an explant tissue, protocols, which are specifically dedicated for ZE and SE, have been developed and successfully used for miRNA analysis [ 28 , 38 , 84 , 95 ].…”