Kunxi Ouyang

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.

Isolation and analysis of α-expansin genes in the tree Anthocephalus chinensis (Rubiaceae).

Expansins are cell wall-associated proteins that induce wall extension and relax stress by disrupting noncovalent bonds between cellulose microfibrils and cross-linking glycan chains, thereby promoting wall creep. Anthocephalus chinensis is a very fast-growing economically important tree found mainly in South Asia. Sixteen cDNAs, designated AcEXPA1 to AcEXPA16 (GenBank accession Nos. FJ417847, JF922686-JF922700) with corresponding genomic DNA sequences (GenBank accession Nos. GQ228823, JF922701-JF922715), were isolated by amplifying conserved domain binding with genomic walking and RACE techniques from four differential growth tissues in A. chinensis. These α-expansin homologues were highly conserved in size and sequence; they had the same sequence structures as an N-terminal signal peptide, three exons and two introns. Their amino acid alignment showed that A. chinensis expansin genes are divided into three subgroups: A, B and C. This study is the first report on expansin genes from A. chinensis. It will be used for a tissue-specific expression model and for studying the relationship between expansin genes, growth rate and wood quality of the xylem in this fast-growing tree.

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.

The complete chloroplast genome of the miracle tree Neolamarckia cadamba and its comparison in Rubiaceae family

Abstract Neolamarckia cadamba is a miracle tree species with considerable economic potential used as a timber wood and traditional medicine resource in South and Southeast Asia. To better understand the molecular basis of its chloroplast biology, we sequenced and characterised the complete chloroplast genome using Illumina pair-end sequencing. The analysis showed a chloroplast genome size of 154,999 bp in length, harbouring a pair of inverted repeats (IRs) of 25,634 bp separated by a large single copy (LSC) sequence of 85,880 bp and a small single copy (SSC) sequence of 17,851 bp. Of the 130 genes present, 96 were unique and 17 were duplicated in the IRs. The coding regions comprised 79 protein genes, 30 tRNA genes and 4 rRNA genes. In the protein-coding genes, nine genes contained one intron each, while another two genes comprised two introns. The overall GC content of the chloroplast genome was 37.6%. Simple sequence repeat (SSR) analysis revealed that most SSRs were A/T-rich, which contributed to the overall AT richness of the N. cadamba chloroplast genome. Additionally, fewer SSRs distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the chloroplast genome. A maximum likelihood phylogenetic analysis showed that N. cadamba was closely related to Morinda officinalis and Emmenopterys henryi, which belong to the Rubiaceae family. This complete chloroplast genome will offer valuable information for the development of highly variable DNA markers for future conservation, genetic engineering studies and variety selection of this miracle tree.