Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin

Background Lupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. The new resources facilitate the in silico identification of candidate genes controlling important agronomic traits. However, a major bottleneck for lupin research and crop improvement is the in planta characterization of gene function. Here, we present an efficient protocol for virus-induced gene silencing (VIGS) to down-regulate endogenous genes in narrow-leafed lupin (NLL) using the apple latent spherical virus (ALSV). Results We identified ALSV as an appropriate VIGS vector able to infect NLL without causing a discernible phenotype. We created improved ALSV vectors to allow for efficient cloning of gene fragments into the viral genome and for easier viral propagation via agroinfiltration of Nicotiana benthamiana. Using this system, we silenced the visual marker gene phytoene desaturase (PDS), which resulted in systemic, homogenous silencing as indicated by bleaching of newly produced tissues. Furthermore, by silencing lysine decarboxylase (LaLDC)—a gene likely to be involved in toxic alkaloid biosynthesis—we demonstrate the applicability of our VIGS method to silence a target gene alone or alongside PDS in a ‘PDS co-silencing’ approach. The co-silencing approach allows the visual identification of tissues where silencing is actively occurring, which eases tissue harvesting and downstream analysis, and is useful where the trait under study is not affected by PDS silencing. Silencing LaLDC resulted in a ~ 61% or ~ 67% decrease in transcript level, depending on whether LaLDC was silenced alone or alongside PDS. Overall, the silencing of LaLDC resulted in reduced alkaloid levels, providing direct evidence of its involvement in alkaloid biosynthesis in NLL. Conclusions We provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.

Transcription Factors in Alkaloid Engineering

Plants produce a large variety of low-molecular-weight and specialized secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used in the pharmaceutical industry. Although alkaloid chemistry has been intensively investigated, characterization of alkaloid biosynthesis, including biosynthetic enzyme genes and their regulation, especially the transcription factors involved, has been relatively delayed, since only a limited number of plant species produce these specific types of alkaloids in a tissue/cell-specific or developmental-specific manner. Recent advances in molecular biology technologies, such as RNA sequencing, co-expression analysis of transcripts and metabolites, and functional characterization of genes using recombinant technology and cutting-edge technology for metabolite identification, have enabled a more detailed characterization of alkaloid pathways. Thus, transcriptional regulation of alkaloid biosynthesis by transcription factors, such as basic helix–loop–helix (bHLH), APETALA2/ethylene-responsive factor (AP2/ERF), and WRKY, is well elucidated. In addition, jasmonate signaling, an important cue in alkaloid biosynthesis, and its cascade, interaction of transcription factors, and post-transcriptional regulation are also characterized and show cell/tissue-specific or developmental regulation. Furthermore, current sequencing technology provides more information on the genome structure of alkaloid-producing plants with large and complex genomes, for genome-wide characterization. Based on the latest information, we discuss the application of transcription factors in alkaloid engineering.

Organ-specific, integrated omics data-based study on the metabolic pathways of the medicinal plant Bletilla striata (Orchidaceae)

Background Bletilla striata is one of the important species belonging to the Bletilla genus of Orchidaceae. Since its extracts have an astringent effect on human tissues, B. striata is widely used for hemostasis and healing. Recently, some other beneficial effects have also been uncovered, such as antioxidation, antiinflammation, antifibrotic, and immunomodulatory activities. As a key step towards a thorough understanding on the medicinal ingredient production in B. striata, deciphering the regulatory codes of the metabolic pathways becomes a major task. Results In this study, three organs (roots, tubers and leaves) of B. striata were analyzed by integrating transcriptome sequencing and untargeted metabolic profiling data. Five different metabolic pathways, involved in polysaccharide, sterol, flavonoid, terpenoid and alkaloid biosynthesis, were investigated respectively. For each pathway, the expression patterns of the enzyme-coding genes and the accumulation levels of the metabolic intermediates were presented in an organ-specific way. Furthermore, the relationships between enzyme activities and the levels of the related metabolites were partially inferred. Within the biosynthetic pathways of polysaccharides and flavonoids, long-range phytochemical transportation was proposed for certain metabolic intermediates and/or the enzymes. Conclusions The data presented by this work could strengthen the molecular basis for further studies on breeding and medicinal uses of B. striata.

Alkaloid Biosynthesis in the Early Stages of the Germination of Argemone mexicana L. (Papaveraceae)

The synthesis of the benzylisoquinoline alkaloids, sanguinarine and berberine, was monitored in Argemone mexicana L. (Papaveracea) throughout the early stages of its hypocotyl and seedling development. Sanguinarine was detected in the cotyledons right after hypocotyl emergence, and it increased continuously until the apical hook unbent, prior to the cotyledonary leaves unfolding, when it abruptly fell. In the cotyledonary leaves, it also remained at low levels. Throughout development, berberine accumulation required the formation of cotyledonary leaves, whereas it was quickly detected in the hypocotyl from the time it emerged. Interestingly, the alkaloids detected in the cotyledons could have been imported from hypocotyls, because no transcriptional activity was detected in there. However, after turning into cotyledonary leaves, important levels of gene expression were noted. Taken together, these results suggest that the patterns of alkaloid tissue distribution are established from very early development, and might require transport systems.

Putative genes in alkaloid biosynthesis identified in Dendrobium officinale by correlating the contents of major bioactive metabolites with genes expression between Protocorm-like bodies and leaves

Background Dendrobium officinale, an endangered Chinese herb, possesses extensive therapeutic effects and contains bioactive ingredients such as major polysaccharides, alkaloids, and minimal flavonoids. We first obtained the protocorm-like bodies (PLBs) of this plant through tissue culture in order to determine the distribution of the main secondary metabolites in each organelle and the PLBs. We then analyzed the correlation between gene expression level from comparative transcriptome sequencing and metabolite content in different organs to identify putative genes encoding enzymes involved in the biosynthesis of polysaccharides, alkaloids, and flavonoids. Results We used seeds as explants for protocorm induction and PLB propagation of D. officinale. The optimal medium formula for PLB propagation was 1/2 MS + α-NAA 0.5 mg·L− 1 + 6-BA 1.0 mg·L− 1 + 2, 4-D 1.5–2.0 mg·L− 1 + potato juice 100 g·L− 1. Stems, PLBs and leaves of D. officinale had the highest content of polysaccharides, alkaloids and flavonoids, respectively. Naringenin was only produced in stem; however, PLBs with high alkaloid content can replace other organs producing alkaloids. The hot water extraction method outperformed the ultrasound-assisted extraction method for extracting polysaccharides from D. officinale. A comparative transcriptome analysis of PLBs and leaves of D. officinale revealed differential expression of genes encoding enzymes involved in polysaccharide, alkaloid and flavonoid biosynthetic pathways. Putative genes encoding enzymes involved in these biosynthetic pathways were identified. Notably, we identified genes encoding the alkaloid biosynthesis enzymes strictosidine β-D-Glucosidase, geissoschizine synthase and vinorine synthase in D. officinale. Conclusions The identification of candidate genes encoding enzymes involved in metabolite biosynthesis will help to explore and protect this endangered species and facilitate further analysis of the molecular mechanism of secondary metabolite biosynthesis in D. officinale.

Root Endophytes and Ginkgo biloba Are Likely to Share and Compensate Secondary Metabolic Processes, and Potentially Exchange Genetic Information by LTR-RTs

Ginkgo biloba is a pharmaceutical resource for terpenes and flavonoids. However, few insights discussed endophytes’ role in Ginkgo, and whether genetic exchange happens between Ginkgo and endophytes remains unclear. Herein, functional gene profiles and repetitive sequences were analyzed to focus on these issues. A total of 25 endophyte strains were isolated from the Ginkgo root and distributed in 16 genera of 6 phyla. Significant morphological diversities lead to the diversity in the COG functional classification. KEGG mapping revealed that endophytic bacteria and fungi potentially synthesize chalcone, while endophytic fungi might also promote flavonoid derivatization. Both bacteria and fungi may facilitate the lignin synthesis. Aspergillus sp. Gbtc_1 exhibited the feasibility of regulating alcohols to lignans. Although Ginkgo and the endophytes have not observed the critical levopimaradiene synthase in ginkgolides synthesis, the upstream pathways of terpenoid precursors are likely intact. The MVK genes in Ginkgo may have alternative non-homologous copies or be compensated by endophytes in long-term symbiosis. Cellulomonas sp. Gbtc_1 became the only bacteria to harbor both MEP and MVA pathways. Endophytes may perform the mutual transformation of IPP and DMAPP in the root. Ginkgo and bacteria may lead to the synthesis and derivatization of the carotenoid pathway. The isoquinoline alkaloid biosynthesis seemed lost in the Ginkgo root community, but L-dopa is more probably converted into dopamine as an essential signal-transduction substance. So, endophytes may participate in the secondary metabolism of the Ginkgo in a shared or complementary manner. Moreover, a few endophytic sequences predicted as Ty3/Gypsy and Ty1/Copia superfamilies exhibited extremely high similarity to those of Ginkgo. CDSs in such endophytic LTR-RT sequences were also highly homologous to one Ginkgo CDS. Therefore, LTR-RTs may be a rare unit flowing between the Ginkgo host and endophytes to exchange genetic information. Collectively, this research effectively expanded the insight on the symbiotic relationship between the Ginkgo host and the endophytes in the root.