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The somatic embryogenic regeneration system is an ideal model system to study the regulation of early developmental processes and morphogenesis in gymnosperms. We have previously generated five larch ( Larix leptolepis ) LaMIR166a overexpression cell lines. The germination rates of mature somatic embryos in transgenic and wild-type (WT) lines were calculated and the results showed that overexpression of the miR166a precursor ( LaMIR166a ) markedly enhanced germination, especially in the a-3, a-4, and a-5 lines. The relative expression of LaMIR166a and miR166a in the LaMIR166a overexpression lines was higher than in the WT control line during the germination process, whereas the expression levels of LaHDZ31–34 increased markedly throughout germination, potentially as a result of feedback regulation of miR166. The effect of miR166a on auxin biosynthesis and signaling genes was also studied. During germination, mRNA levels of Nitrilase ( LaNIT ), Auxin response factor1 ( LaARF1 ), and LaARF2 were markedly higher in LaMIR166a overexpressing lines. These results indicated that indole-3-acetic acid (IAA) synthesis is required for germination in L. leptolepis . Further exogenous application of IAA at different concentrations showed that 2 mg L −1 IAA clearly promoted germination, resulting in a 56% germination rate for L. leptolepis somatic embryos. This shows that IAA plays a vital role in controlling the germination ability of someatic embryos in L. leptolepis . Our results suggest that miR166a and LaHDZ31–34 have important roles in auxin biosynthesis and signaling during the germination of somatic embryos in L. leptolepis .

Somatic embryo (SE) regeneration is an ideal experimental system to realize rapid propagation of excellent clones and genetic improvement for perennial gymnosperms. In the present study, genes encoding the miRNA166 precursor were identified and LamiR166a was successfully transformed into the gymnosperm Larix leptolepis (L. leptolepis) and five LamiR166a over-expressed embryonic cell lines were screened out as stable embryo masses. As expected, the targets of miR166a, LaHDZ31-34, were all down-regulated in transgenic lines according to qRT-PCR results. The results showed that the percentage of normal SEs with 4–7 cotyledons was 77.0 % in wild type (WT) lines, but was reduced to 60.3 % in the pSuper::MIR166a lines with “cup-shaped” embryos comprised 7.0 % of WT and 20.7 % of transgenic embryos. Microscopic observation further showed that the intermediate region surrounded by the cotyledons was larger than in the control, with no upward bulge of the shoot apical meristem (SAM). The expression pattern of the two meristem marker genes CLAVATA (CLV) and WUSCHEL-related homeobox (WOX) were investigated. The results showed that the expression levels of WOX were three times higher in transgenic lines than in WT samples, which suggest that miR166a may indirectly regulate SAM development by directly affecting WOX expression. Besides, overexpression of LamiR166a clearly increased the rooting rate and promoted lateral root formation in L. leptolepis seedlings. These results may provide new insights into the regulatory role of miR166 in gymnosperms, and also new applications for forestry production in practice.

Somatic embryogenesis (SE) involves complex molecular signalling pathways. Understanding molecular mechanism of SE in Larix leptolepis (L. leptolepis) can aid research on genetic improvement of gymnosperms. Previously, we obtained five LaMIR166a (miR166a precursor) -overexpression embryonic cell lines in the gymnosperm Larix leptolepis. The proliferation rates of pro-embryogenic masses in transgenic and wild-type lines were calculated. Overexpression of the miR166a precursor LaMIR166a led to slower proliferation. When pro-embryogenic masses were transferred to maturation medium, the relative expression of LaMIR166a and miR166a in the LaMIR166a-overexpression lines was higher than in the wild-type during SE, while LaHDZ31–34 expression levels also increased without negative control by miR166, suggesting that regulation of HD-ZIP III by miR166a exits stage-specific characteristics. The key indole-3-acetic acid (IAA) biosynthetic gene Nitrilase of L. leptolepis (LaNIT) was identified and the effects of miR166a on auxin biosynthesis and signalling genes were studied. During SE, LaNIT, Auxin response factor1 (LaARF1) and LaARF2 mRNA levels and IAA contents were markedly higher in LaMIR166a-overexpression lines, which revealed lower deformity rate of embryos, indicating endogenous IAA synthesis is required for somatic embryo maturation in L. leptolepis. Additionally, the IAA biosynthesis and signalling genes showed similar expression patterns to LaHDZ31–34, suggesting HD-ZIP III genes have a positive regulatory effect on LaNIT. Our results suggest miR166a and LaHDZ31–34 have important roles in auxin biosynthesis and signalling during SE, which might determine if the somatic embryo normally developed to mature in L. leptolepis.

Somatic embryogenesis is an ideal model process for studying early plant development. Embryonic cell lines of Larix kaempferi (Lamb.) Carr overexpressing LaMIR166a were obtained in our previous study. Here, a combination of de novo transcriptomics and extensively targeted metabolomics was used to study the transcriptional profiles and metabolic changes in wild-type and LaMIR166a-overexpressed embryonic cell lines. A total of 459 metabolites were found in the wild-type and transgenic cell lines. Compared to those in the wild-type cell lines, transcripts and metabolites were significantly altered in the LaMIR166a-overexpressed cell lines. Among differentially expressed genes (DEGs), phenylalanine and flavonoid synthesis genes were significantly enriched, and among differentially accumulated metabolites (DAMs), phenolic acids and flavonoids accumulated in particularly high amounts. Thus, the flavonoid biosynthetic pathway seems to be the most abundant pathway in response to LaMIR166a overexpression. Based on the Kyoto Encyclopedia of Genes and Genomes database, the association analysis of metabolome and transcriptome data showed that flavonoid biosynthesis and plant hormone signal transduction processes were significantly changed in miR166a-overexpression lines, suggesting that miR166 might be involved in these processes. The present study identified a number of potential metabolites associated with LaMIR166a overexpression, providing a significant foundation for a better understanding of the regulatory mechanisms underlying miR166.

The study of somatic embryogenesis can provide insight into early plant development. We previously obtained LaMIR166a-overexpressing embryonic cell lines of Larix kaempferi (Lamb.) Carr. To further elucidate the molecular mechanisms associated with miR166 in this species, the transcriptional profiles of wild-type (WT) and three LaMIR166a-overexpressing transgenic cell lines were subjected to RNA sequencing using the Illumina NovaSeq 6000 system. In total, 203,256 unigenes were generated using Trinity de novo assembly, and 2467 differentially expressed genes were obtained by comparing transgenic and WT lines. In addition, we analyzed the cleaved degree of LaMIR166a target genes LaHDZ31–34 in different transgenic cell lines by detecting the expression pattern of LaHdZ31–34, and their cleaved degree in transgenic cell lines was higher than that in WT. The downstream genes of LaHDZ31–34 were identified using Pearson correlation coefficients. Yeast one-hybrid and dual-luciferase report assays revealed that the transcription factors LaHDZ31–34 could bind to the promoters of LaPAP, LaPP1, LaZFP5, and LaPHO1. This is the first report of gene expression changes caused by LaMIR166a overexpression in Japanese larch. These findings lay a foundation for future studies on the regulatory mechanism of miR166.