Comparisons of different RNA extraction methods on woody tissues of the tropical tree, Aquilaria malaccensis

Aquilaria malaccensis (karas) produces oleoresin in its wood (known as agarwood) when responding to wounding and microbial infection. Wood tissues are known to contain high levels of polysaccharides, polyphenolics and secondary metabolites, which make RNA extraction challenging. In this work, six different methods for extracting RNA from wood tissues of A. malaccensis were compared. RNA yield, purity, and integrity number, were used as parameters to evaluate the efficiency of each method. Conventional methods yielded RNA with good purity but the RNA integrity was poor. The commercial RNeasy Plant Mini kit protocol was modified by means of scaling-up the reaction and combining all aliquots in the same RNeasy spin column, and yielded the highest yield while maintaining the integrity of the RNA. We found that this kit with some modifications was most suitable for extracting RNA from healthy wood and agarwood. This study is essential for future molecular studies on agarwood

Transcriptome reveals senescing callus tissue of Aquilaria malaccensis, an endangered tropical tree, triggers similar response as wounding with respect to terpenoid biosynthesis

Aquilaria malaccensis is an endangered tree species listed in the Appendix II of CITES. It is a main source of highly valuable resinous wood known as agarwood, which is rich in secondary metabolites. De novo assembly of sequences produced by transcriptome sequencing using next-generation sequencing technologies offers a rapid approach to obtain expressed gene sequences for non-model organisms such as Aquilaria. To investigate the genes and pathways that might control molecular mechanism of A. malaccensis under controlled environment, we sequenced two transcriptome libraries constructed from mRNAs of healthy and senescing callus tissues using Illumina sequencing. We obtained 200,062,275 and 166,544,202 reads for healthy and senescing callus libraries, respectively. We compiled 231,594 transcripts and identified 107,593 transcripts by similarity analysis against the National Center for Biotechnology Information (NCBI) public database. A total of 96,743 transcripts were functionally annotated using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. We assigned 46,076 of the transcripts to a total of 144 KEGG pathways. We focused on genes identified as contributing to fragrant compound synthesis and defense response pathways, which are important pathways leading to agarwood compound formation. This study provides abundant transcriptomic data and valuable sequence resources for future genomic studies on A. malaccensis. This is the first report of callus transcriptome from A. malaccensis

Temporal expression of a putative homogentisate solanesyltransferase cDNA in wounded Aquilaria malaccensis, an endangered tropical tree

Homogentisate prenytransferase (HPI) generally catalyses prenylation reactions in tocochromanol and plastoquinone-9 biosynthesis, while homogentisate solanesyltransferase (HST) is specific to reaction leading to plastoquinone, an essential component in the synthesis of carotenoid, a powerful antioxidant and precursor to vitamin A. In Aquilaria spp. abiotic stress in the form of wounding is the main trigger for the production of a highly-valued terpene-rich wood known as agarwood. Putative HST cDNA, AmHST1 was cloned from total RNA of callus tissue of Aquilaria malaccensis using reverse transcription approach. Based on a partial HST sequence, specific primers were initially designed to amplify the internal open reading frame region followed by RACE, which successfully amplified the cDNA. The partial length AmHST1 cDNA measured about 1182 bp nucleotides and encodes a polypeptide of 392 amino acid. Sequence alignment revealed that AmHST1 shares 74% - 77% similarity with HPT from Arabidopsis and Theobroma cacao. Gene expression analysis indicated that the AmHST1 expression was suppressed in wounded tissues. Results suggest that there should be a potential trade-off between genes involved in plastoquinone and terpenoid synthesis as they both share similar upstream genes and precursors. When facing a major abiotic stress such as wounding, the latter is favoured

De novo assembly, annotation and analysis of transcriptome sequences of callus culture from Aquilaria malaccensis Lam.

Aquilaria malaccensis is a major source of agarwood, a rare and highly priced wood product. Due to its high demand, it is endangered and listed in the Appendix II of Convention on International Trade in Endangered Species of Wild Fauna and Flora. Because of insufficient genomic and transcriptomic data available in public database for understanding the molecular basis of agarwood formation, the goal of this study was to obtain A. malaccensis expressed gene sequences using the transcriptome sequencing by next-generation sequencing (NGS). To obtain sufficient data from NGS sequencing, a high quality RNA sample is required. The RNA yield, purity, and integrity of six different extraction methods were compared. Conventional methods yielded RNA with good purity but the RNA integrity was poor. When using modified RNeasy Plant Mini kit, the highest yield was obtained while maintaining the integrity of RNA. This method was used to extract total RNA from callus samples and sent for transcriptome sequencing. Callus tissues were treated under stress condition by nutrient shortage of 1.1 μm 1- naphthaleneacetic acid (NAA), 2.2 μm 6-benzylaminopurine (BAP), and 15 g/L sucrose; collected during death phase when it producing brownish exudates and agarwood scent. Two cDNA libraries were constructed from mRNAs of untreated and treated callus tissues and sequenced using paired-end libraries on an Illumina HiSeq2000 platform. After filtering and trimming, a total of 200,062,275 and 166,544,202 clean reads were obtained for untreated and treated callus libraries, respectively. De novo assembly was carried out using SOAPdenovo-Trans and TGICL. A total of 231,594 unigenes were generated from the assembly. Assembled sequences were annotated using BLASTX against the NCBI non-redundant protein database where 41.5% unigenes showed significant alignment. The differential gene expression value between untreated and treated samples were calculated using DEGseq. A total of 14,029 genes were identified as differentially expressed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were reported using BLAST2GO software. Out of the 107,593 unigenes that showed significant alignment with non-redundant protein database, 96,743 unigenes were successfully annotated with at least one GO term. The annotations were classified into the three main GO categories: biological processes (50.7%), molecular functions (24.0%) and cellular components (25.3%). A total of 144 KEGG pathways were identified from 46,076 unigenes. Enrichment analysis showed that the genes were enriched in the stress responses and sesquiterpene biosynthesis activity. This is the first comprehensive de novo transcriptome assembly and profiling for A. malaccensis. These transcriptome libraries provide valuable transcriptomic reference resource for future research

Genes associated to wounding from Aquilaria malaccensis

Agarwood is an aromatic oleoresin produced by Aquilaria in response to wounding and biological attack but the molecular mechanism is unknown. In this study, several candidate genes in wounding and defense responses were cloned and identified. Deduced amino acid of the cloned sequences exhibited sequence similarities to their respective homologs: transcription factors of the WRKY gene family (AmWRKY) and β-1,3-glucanase (AmGLU). Phenylalanine ammonia-lyase (AmPAL) from previous work was also included. A wounding-stress experiment revealed their expression profiles via qRT-PCR analysis. AmWRKY was induced early after wounding (3 hours), while AmPAL and AmGLU expressions were induced later (16 hours). It is possible that AmWRKY mediates early wounding response while AmPAL mediates response to fungal infection by co-inducing AmGLU