Juncheng Li

Biochemical and molecular changes associated with heteroxylan biosynthesis in Neolamarckia cadamba (Rubiaceae) during xylogenesis

Wood, derived from plant secondary growth, is a commercially important material. Both cellulose and lignin assembly have been well studied during wood formation (xylogenesis), but heteroxylan biosynthesis is less well defined. Elucidation of the heteroxylan biosynthetic pathway is crucial to understand the mechanism of wood formation. Here, we use Neolamarckia cadamba, a fast-growing tropical tree, as a sample to analyze heteroxylan formation at the biochemical and molecular levels during wood formation. Analysis of the non-cellulosic polysaccharides isolated from N. cadamba stems shows that heteroxylans dominate non-cellulosic polysaccharides and increase with xylogenesis. Microsomes isolated from stems of 1-year-old N. cadamba exhibited UDP-Xyl synthase and xylosyltransferase activities with the highest activity present in the middle and basal stem regions. To further understand the genetic basis of heteroxylan synthesis, RNA sequencing (RNA-seq) was used to generate transcriptomes of N. cadamba during xylogenesis. The RNA-seq results showed that genes related to heteroxylan synthesis had higher expression levels in the middle and basal part of the stem compared to the apical part. Our results describe the heteroxylan distribution and heteroxylan synthesis trait in N. cadamba and give a new example for understanding the mechanism of heteroxylan synthesis in tropical tree species in future.

A simple method for RNA isolation from various tissues of the tree Neolamarckia cadamba.

Plant tissues contain abundant polysaccharides, phenolic compounds and other metabolites, which makes it difficult to isolate high-quality RNA from them. In addition, Neolamarckia cadamba contains large quantities of other components, particularly RNA-binding alkaloids, which makes the isolation even more challenging. Here, we describe a concise and efficient RNA isolation method that combines the cetyltrimethyl ammonium bromide (CTAB) and Plant RNA Kit (Omega) protocols. Gel electrophoresis showed that RNA extracted from all tissues, using this protocol, was of good integrity and without DNA contamination. Furthermore, the isolated RNA was of high purity, with an A260/A280 ratio of 2.1 and an A260/A230 ratio of >2.0. The isolated RNA was also suitable for downstream applications, such as reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR). The RNA isolation method was also efficient for recalcitrant plant tissues.

Transcriptomic Analysis of Multipurpose Timber Yielding Tree Neolamarckia cadamba during Xylogenesis Using RNA-Seq

Neolamarckia cadamba is a fast-growing tropical hardwood tree that is used extensively for plywood and pulp production, light furniture fabrication, building materials, and as a raw material for the preparation of certain indigenous medicines. Lack of genomic resources hampers progress in the molecular breeding and genetic improvement of this multipurpose tree species. In this study, transcriptome profiling of differentiating stems was performed to understand N. cadamba xylogenesis. The N. cadamba transcriptome was sequenced using Illumina paired-end sequencing technology. This generated 42.49 G of raw data that was then de novo assembled into 55,432 UniGenes with a mean length of 803.2bp. Approximately 47.8% of the UniGenes (26,487) were annotated against publically available protein databases, among which 21,699 and 7,754 UniGenes were assigned to Gene Ontology categories (GO) and Clusters of Orthologous Groups (COG), respectively. 5,589 UniGenes could be mapped onto 116 pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Among 6,202 UniGenes exhibiting differential expression during xylogenesis, 1,634 showed significantly higher levels of expression in the basal and middle stem segments compared to the apical stem segment. These genes included NAC and MYB transcription factors related to secondary cell wall biosynthesis, genes related to most metabolic steps of lignin biosynthesis, and CesA genes involved in cellulose biosynthesis. This study lays the foundation for further screening of key genes associated with xylogenesis in N. cadamba as well as enhancing our understanding of the mechanism of xylogenesis in fast-growing trees.

Functional identification of an EXPA gene (NcEXPA8) isolated from the tree Neolamarckia cadamba

ABSTRACT As a class of important cell growth regulators, expansins have been studied for over 20 years. Since Neolamarckia cadamba (Roxb.) Bosser was praised as a ‘miraculous tree’ at the World Forestry Congress in 1972 due to its rapid growth. A lot of research has been carried out to uncover the underlying mechanisms of this rapid growth. Based on previous findings and our research, we hypothesized that expansins may play an important role in such growth. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the N. cadamba expansin family member NcEXPA8 is highly expressed in all four young tissues, particularly in the cambium region, suggesting that NcEXPA8 acts as a key regulator of secondary growth in this gene family. Overexpression of NcEXPA8 in Arabidopsis thaliana increased the diameter and height of the main stem. It also promoted interfascicular fiber cell elongation and cell-wall thickness but did not alter the cellulose content in the cell wall. These results suggested that expansins act as activators during secondary fiber cell elongation during tip growth to promote plant growth.

Screening Reliable Reference Genes for RT-qPCR Analysis of Gene Expression in Moringa oleifera.

Moringa oleifera is a promising plant species for oil and forage, but its genetic improvement is limited. Our current breeding program in this species focuses on exploiting the functional genes associated with important agronomical traits. Here, we screened reliable reference genes for accurately quantifying the expression of target genes using the technique of real-time quantitative polymerase chain reaction (RT-qPCR) in M. oleifera. Eighteen candidate reference genes were selected from a transcriptome database, and their expression stabilities were examined in 90 samples collected from the pods in different developmental stages, various tissues, and the roots and leaves under different conditions (low or high temperature, sodium chloride (NaCl)- or polyethyleneglycol (PEG)- simulated water stress). Analyses with geNorm, NormFinder and BestKeeper algorithms revealed that the reliable reference genes differed across sample designs and that ribosomal protein L1 (RPL1) and acyl carrier protein 2 (ACP2) were the most suitable reference genes in all tested samples. The experiment results demonstrated the significance of using the properly validated reference genes and suggested the use of more than one reference gene to achieve reliable expression profiles. In addition, we applied three isotypes of the superoxide dismutase (SOD) gene that are associated with plant adaptation to abiotic stress to confirm the efficacy of the validated reference genes under NaCl and PEG water stresses. Our results provide a valuable reference for future studies on identifying important functional genes from their transcriptional expressions via RT-qPCR technique in M. oleifera.

Evolution and expression patterns of the trehalose-6-phosphate synthase gene family in drumstick tree (Moringa oleifera Lam.)

Main conclusionMoringa oleifera TPSs were genome-wide identified for the first time, and a phylogenetic analysis was performed to investigate evolutionary divergence. The qRT-PCR data show that MoTPS genes response to different stress treatments.The trehalose-6-phosphate synthase (TPS) family is involved in a wide range of stress-resistance processes in plants. Its direct product, trehalose-6-phosphate, acts as a specific signal of sucrose status and a regulator to modulate carbon metabolism within the plant. In this study, eight TPS genes were identified and cloned based on the M. oleifera genome; only MoTPS1 exhibited TPS activity among Group I proteins. The characteristics of the MoTPS gene family were determined by analyzing phylogenetic relationships, gene structures, conserved motifs, selective forces, and expression patterns. The Group II MoTPS genes were under relaxed purifying selection or positive selection. The glycosyltransferase family 20 domains generally had lower Ka/Ks ratios and nonsynonymous (Ka) changes compared with those of trehalose-phosphatase domains, which is consistent with stronger purifying selection due to functional constraints in performing TPS enzyme activity. Phylogenetic analyses of TPS proteins from M. oleifera and 17 other plant species indicated that TPS were present before the monocot–dicot split, whereas Group II TPSs were duplicated after the separation of dicots and monocots. Quantitative real-time PCR analysis showed that the expression patterns of TPSs displayed group specificities in M. oleifera. Particularly, Group I MoTPS genes closely relate to reproductive development and Group II MoTPS genes closely relate to high temperature resistance in leaves, stem, stem tip and roots. This work provides a scientific classification of plant TPSs, dissects the internal relationships between their evolution and expressions, and promotes functional researches.

Phylogeographic pattern suggests a general northeastward dispersal in the distribution of Machilus pauhoi in South China

Machilus pauhoi Kanehira is an important timber species in China. A provenance trial was recently set up to evaluate the growth performance of trees from different localities, with the aim of designing seed transfer guidelines. Here, we tested twelve nuclear microsatellite markers derived from other species of the Lauraceae family and investigated population genetic structure in M. pauhoi. Both the number of observed alleles per locus (Na) and the polymorphic information content (PIC) significantly decreased against the latitude, but showed an insignificant decrease against the longitude. Heterozygosity (Ho) and gene diversity (h) exhibited a weak correlation with geographic location. Private alleles were present in multiple populations, and a moderate level of population genetic differentiation was detected (Gst = 0.1691). The joint pattern of genetic diversity (Na, PIC, Ho, and h) suggests that general northeastward dispersal led to the current distribution of M. pauhoi. Significant but weak effects of isolation-by-distance (IBD) occurred, implicating the mountain ranges as the major barrier to gene flow. Both STRUCTURE and hierarchical clustering analyses showed three distinct groups of populations related to the physical connectivity among mountain ranges. A priority in designing genetic conservation should be given to the populations at the southwest side of the species’ distribution. This conservation strategy can also be combined with the pattern of adaptive genetic variation from the provenance trial for comprehensive genetic resource management of native M. pauhoi.