Genome-wide analysis reveals dynamic changes in expression of microRNAs during vascular cambium development in Chinese fir, Cunninghamia lanceolata

Abstract

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that play key roles in the process of plant development. To date, extensive studies of miRNAs have been performed in a few model plants, but few efforts have focused on small RNAs (sRNAs) in conifers because of the lack of reference sequences for their enormous genomes. In this study, Solexa sequencing of three sRNA libraries obtained from dormant, reactivating, and active vascular cambium in Chinese fir (Cunninghamia lanceolata) using tangential cryosectioning identified 20 known miRNA families and 18 novel potential miRNAs, of which nine novel miRNA precursors were validated by RT-PCR and sequencing. More than half of these novel miRNAs displayed stage-specific expression patterns in the vascular cambium. Furthermore, analysing the 103 miRNAs and their predicted targets indicated that about 70% appeared to negatively regulate their targets, of which two target genes involved in the regulation of cambial cell division were validated via RNA ligase-mediated rapid amplification of 5'-cDNA ends (RLM 5'-RACE) and transient co-expression in Nicotiana benthamiana leaves. Interestingly, miRNA156 and miRNA172 may regulate the phase transition in vascular cambium from dormancy to active growth. These results provide new insights into the important regulatory functions of miRNAs in vascular cambium development and wood formation in conifers.

Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR

“…Another common HKG, EF1a , was validated as one of the best choices for Brachiaria brizantha [54]. In previous studies, scientists have applied and identified HKGs as internal control genes in different tissues of Chinese fir, including leaves, shoot apical meristems, stems, barks, xylems and vascular cambiums [4,5,55,56]. However, identification of HKGs in Chinese fir seedlings upon different abiotic stresses has not been reported.…”

“…Chinese fir is a fast-growing conifer that belongs to family Cupressaceae, and is a commercially important tree of the lumber, pulp, and paper industry. Biological studies have recently revealed the molecular mechanism underlying the control of the developmental processes and physiological responses of the Chinese fir [4,5]. …”

“…Early studies investigated the effects of growth conditions on its morphological characters and biochemical components [3,6]. Using advanced genetic approaches, researchers have recently focused on elucidating the molecular mechanisms underlying the regulation of physiological responses, including analyses of gene function [7,8], transcriptome [4,9], or small non-coding RNA and miRNA identification [5,10]. Although next-generation sequencing (NGS) technologies allow ultrahigh-throughput and highly accurate quantification of gene expression levels, the associated high cost of sequencing and post-verification on extensive amounts of nucleic acids (DNA or RNA) limit its widespread use [11,12,13].…”

Transcriptional regulation of vascular cambium activity during the transition from juvenile to mature stages in Cunninghamia lanceolata

“…Briefly, 30 m sections were isolated by tangential cryosectioning at −24 • C with a Leica CM1850 Cryostat (Leica Microsystems Nussloch GmbH, Germany) equipped with a steel knife. Transverse sections taken from both ends of the specimen and stained with aniline blue were used to locate the position of tangential sections, as described previously (Qiu et al, 2015).…”

“…During the past decades, millions of C. lanceolat trees have been planted and more areas are being re-planted as second or later rotation (Zhang et al, 2004). Based on our previous investigations in C. lanceolata (Wan et al, 2012;Qiu et al, 2013;Qiu et al, 2015), we further examined the transcriptome profile of cambial meristem at these three developmental stages, combined with the morphological analysis. To the best of our knowledge, this study is the first to characterize the transcriptome dynamics of coniferous trees during the transition from juvenile to mature stages, which then provides new insights into the molecular mechanisms underlying vascular cambium development and wood production.…”