MdMYB4 enhances apple callus salt tolerance by increasing <Emphasis Type="Italic">MdNHX1</Emphasis> expression levels

Here, we performed comparative miRNA profiling in wild type and early flowering transgenic Chrysanthemum morifolium with constitutive expression of APETALA1 (AP1)-like gene, HAM92 (Helianthus annuus). Six sRNA libraries constructed from leaves and shoot apexes after the short day photoperiod initiation, as well as from opened inflorescence after anthesis were sequenced and analyzed. A total of 324 members (163 families) of putative conserved miRNAs and 30 candidate novel miRNAs specific for C. morifolium (cmo-miRNAs) were identified. Bioinformatic analysis revealed 427 and 138 potential mRNA targets for conserved and novel cmo-miRNAs, respectively. These genes were described in Gene Ontology terms and found to be implicated in a broad range of signaling pathways. Plant- and tissue-specific expression of 9 highly conserved cmo-miRNAs was compared between wild type and transgenic chrysanthemum lines with ectopic expression of AP1-like genes HAM92 and CDM111 (C. morifolium), using RT-qPCR and cmo-miR162a as a reference miRNA. The results of our study provide a framework for further investigation of miRNA evolution and functions in higher plants, as well as their roles in flowering control.     

Identification of novel miRNAs and their target genes in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

Despite Eucalyptus grandis being the most widely planted hardwood tree globally, along with the availability of a sequenced genome and easily accessible functional genetic tools, the quantities and roles of miRNA in its developmental processes remains largely unknown. In this study, we constructed small RNA libraries by high-throughput sequencing from Eucalyptus grandis samples, and 386 novel miRNAs were identified by miRDeep2. We found 179 novel miRNAs, 41 miRNA families, and 456 target genes in leaf samples, and 257 novel miRNAs, 61 miRNA families, and 483 target genes in stem samples. The function of the MIR396 family of miRNAs in Eucalyptus grandis was found to be mainly associated with the process of cell growth. By annotation analysis of miRNA targets, we found that some target genes, such as GRF, expansin-A15, and RPS2, had a close correlation in stem. Finally, the three randomly selected members of the MIR396 family were confirmed to express in Eucalyptus grandis by qRT-PCR, indicating that our reported miRNAs were existed. The identification of miRNAs and their target genes will lead to a greater understanding of the role of miRNAs in the physiology, growth, and development of Eucalyptus grandis trees.     

The use of miRNAs as reference genes for miRNA expression normalization during <Emphasis Type="Italic">Lilium</Emphasis> somatic embryogenesis by real-time reverse transcription PCR analysis

Expression profiling of miRNAs has the ability to reveal the essence of somatic embryogenesis (SE). qRT-PCR is one of the most commonly used techniques for dynamic miRNA detection but requires optimal reference genes for data reliability. This is the first report on reference gene validation for miRNA expression normalization in Lilium (Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor). In this study, seventeen miRNAs together with two snRNAs (U4, U6), one rRNA (5S rRNA) and three protein-coding genes (FP, ACT, GAPDH) were selected as reference candidates, and their expression stability was validated by qRT-PCR among eleven developing SE cultures in two lilies. Four normalization algorithms, including geNorm, BestKeeper, NormFinder and RefFinder, were also used to evaluate the stability of the reference candidates. For Lilium pumilum DC. Fisch., lpu-miR159a was the optimal reference gene during SE, followed by lpu-miR408b, while U6 was the least stable reference candidate. For Lilium davidii var. unicolor, FP presented greater stability than did half of the miRNA candidates, but the best reference gene was lda-miR162, followed by lda-miR159a. Further analysis of the expression level of miR156 and miR529 was used to evaluate the validity of the reference genes in both lilies. In general, miRNAs are superior to common protein-coding genes and snRNAs / rRNAs as reference genes for miRNA expression normalization during Lilium SE, and the most suitable reference miRNA is different between two species in the same Lilium genus. This is a pioneer study using suitable miRNAs as reference genes in Lilium and constitutes a small but essential step for the further exploration of miRNA function in Lilium, thus offering valuable references for other plants.     

Comparative analysis of microRNAs and putative target genes in hybrid clone <Emphasis Type="Italic">Paulownia</Emphasis> ‘yuza 1’ under drought stress

Drought stress in plants often leads to reduced productivity and limited geographic distribution, which can affect human life and ecosystems. The responses of diploid and tetraploid Paulownia tomentosa × Paulownia fortunei to drought have been reported, but the effects of drought stress on the levels of microRNA (miRNA) expression have not been published so far. Here, we constructed four small RNA (sRNA) libraries and four corresponding degradome libraries of well-watered and severe drought-treated diploid and tetraploid plants to identify the miRNAs and their putative target genes in Paulownia ‘yuza 1’, a P. tomentosa × P. fortunei hybrid clone, by sRNA and degradome sequencing. The putative target genes of miRNAs were annotated with gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. Three conserved and 21 novel miRNAs responsive to drought stress were found, in which 15 were identified as the main drought responsive miRNAs that conferred higher resistance in tetraploid than in diploid of Paulownia ‘yuza 1’. Our results will lay the foundation for investigating the roles of miRNAs in Paulownia and other trees in response to drought.     

Deep sequencing discovery and profiling of conserved and novel miRNAs in the ovule of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Key message

High-throughput sequencing and subsequent analysis identified multiple miRNAs closely related to ovule, indicating that miRNAs are important in Ginkgo biloba ovule.

Abstract

MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs that play crucial regulatory roles in the process of plant growth and development. However, limited information regarding their functions in gymnosperm reproduction is available. Here, we used high-throughput sequencing combined with computational analysis to identify and characterize miRNAs from ovules of G. biloba, and identified 34 conserved miRNA families and 99 novel miRNAs. The precursor sequences of several of the conserved and novel miRNAs were further validated by RT-PCR and sequencing. Furthermore, we found that some target genes, e.g. MYB, homeodomain-leucine zipper (HD-ZIPIII) and auxin response factor (ARF), may be involved in ovule development, and that the significantly enriched pathways of some miRNA targets were related to plant–pathogen interactions and the biosynthesis of secondary metabolites. Twenty-six conserved miRNA families were found to be expressed in both leaves and ovules, while miRNA156, miRNA164, miRNA167, miRNA169, miRNA172 and miRNA390 were up-regulated in ovules. Thus, multiple miRNAs closely related to G. biloba ovule development were identified, resulting in a greater understanding of the important regulatory functions of miRNAs in plant ovules.