Expression of Beta-Glucosidase Increases Trichome Density and Artemisinin Content in Transgenic Artemisia Annua Plants

Artemisinin is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. However, a low level of accumulation of artemisinin in Artemisia annua is a major limitation for its production and delivery to malaria endemic areas of the world. While several strategies to enhance artemisinin have been extensively explored, enhancing storage capacity in trichome has not yet been considered. Therefore, trichome density was increased with the expression of β glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation. Transgene (bgl1) integration and transcript was confirmed by molecular analysis. Trichome density increased up to 20% in leaves and 66% in flowers of BGL1 transgenic plants than Artemisia control plants. High-performance liquid chromatography (HPLC, MS-TOF) data showed that artemisinin content increased up to 1.4% in leaf and 2.56% in flowers (g-1DW), similar to the highest yields achieved so far through metabolic engineering. Artemisinin was enhanced up to 5-fold in BGL1 transgenic flowers. The present study opens the possibility of increasing artemisinin content by manipulating trichomes density, which is a major reservoir of artemisinin. Combining biosynthetic pathway engineering with enhancing trichome density may further increase artemisinin yield in A. annua. Because oral feeding of Artemisia plant cells reduced parasitemia more efficiently than the purified drug, reduced drug resistance and cost of prohibitively expensive purification process, enhanced expression should play a key role in making this valuable drug affordable to treat malaria in a large global population that disproportionally impacts low-socioeconomic areas and underprivileged children

Complete Chloroplast Genome Sequence of an Orchid Model Plant Candidate: Erycina pusilla Apply in Tropical Oncidium Breeding

Oncidium is an important ornamental plant but the study of its functional genomics is difficult. Erycina pusilla is a fast-growing Oncidiinae species. Several characteristics including low chromosome number, small genome size, short growth period, and its ability to complete its life cycle in vitro make E. pusilla a good model candidate and parent for hybridization for orchids. Although genetic information remains limited, systematic molecular analysis of its chloroplast genome might provide useful genetic information. By combining bacterial artificial chromosome (BAC) clones and next-generation sequencing (NGS), the chloroplast (cp) genome of E. pusilla was sequenced accurately, efficiently and economically. The cp genome of E. pusilla shares 89 and 84% similarity with Oncidium Gower Ramsey and Phalanopsis aphrodite, respectively. Comparing these 3 cp genomes, 5 regions have been identified as showing diversity. Using PCR analysis of 19 species belonging to the Epidendroideae subfamily, a conserved deletion was found in the rps15-trnN region of the Cymbidieae tribe. Because commercial Oncidium varieties in Taiwan are limited, identification of potential parents using molecular breeding method has become very important. To demonstrate the relationship between taxonomic position and hybrid compatibility of E. pusilla, 4 DNA regions of 36 tropically adapted Oncidiinae varieties have been analyzed. The results indicated that trnF-ndhJ and trnH-psbA were suitable for phylogenetic analysis. E. pusilla proved to be phylogenetically closer to Rodriguezia and Tolumnia than Oncidium, despite its similar floral appearance to Oncidium. These results indicate the hybrid compatibility of E. pusilla, its cp genome providing important information for Oncidium breeding

Arabidopsis Tic40 Expression In Tobacco Chloroplasts Results In Massive Proliferation Of The Inner Envelope Membrane And Upregulation Of Associated Proteins

The chloroplast inner envelope membrane (IM) plays essential roles in lipid synthesis, metabolite transport, and cellular signaling in plants. We have targeted a model nucleus-encoded IM protein from Arabidopsis thaliana, pre-Tic40-His, by relocating its expression from the nucleus to the chloroplast genome. Pre-Tic40-His was properly targeted, processed, and inserted. It attained correct topology and was folded and assembled into a TIC complex, where it accounted for up to 15% of the total chloroplast protein. These results confirm the existence of a novel pathway for protein targeting to the IM. Tic40-His overexpression resulted in a massive proliferation of the IM (up to 19 layers in electron micrographs) without significant effects on plant growth or reproduction. Consistent with IM proliferation, the expression levels of other endogenous IM proteins (IEP37, PPT, Tic110) were significantly (10-fold) upregulated but those of outer envelope membrane (Toc159), stromal (hsp93, cpn60), or thylakoid (LHCP, OE23) proteins were not increased, suggesting retrograde signal transduction between chloroplast and nuclear genomes to increase lipid and protein components for accommodation of increased accumulation of Tic40. This study opens the door for understanding the regulation of membrane biogenesis within the organelle and the utilization of transgenic chloroplasts as bioreactors for hyperaccumulation of membrane proteins for biotechnological applications. © 2008 American Society of Plant Biologists

Variations in 5 regions in 19 Epidendroideae species.

<p>Numbers indicate the positions of the chloroplast genome. The angled dashed lines indicate deletions. The green triangle indicates an insertion. The yellow areas indicate diverse sequences. ¹including <i>Calanthe discolor</i> and <i>Calanthe sylvatica</i>; ²including <i>Geodorum densiflorum</i>; ³including <i>Cymbidium aloifolium</i> and <i>Geodorum densiflorum</i>.</p

Gene map of <i>Erycina pusilla</i> chloroplast genome.

<p>Genes on the outside of the map are transcribed clockwise whereas genes on the inside of the map are transcribed counterclockwise. Colors indicate genes with different functional groups.</p

Phylogenetic analysis of 36 Oncidiinae species.

<p>These trees are based on the nucleotide sequences of A. <i>trnF</i>-<i>ndhJ</i>, B. <i>trnF</i>-<i>ndhJ</i> and <i>trnH</i>-<i>psbA</i> cpDNA regions. The numbers indicate bootstrap probability values. The names of genera are abbreviated as follows: <i>Bllra</i>., <i>Beallara</i>; <i>Comp</i>., <i>Comparettia</i>; <i>Dgmra</i>., <i>Degarmoara</i>; <i>Incdm</i>., <i>Ionocidium</i>; <i>Mac</i>., <i>Macradenia</i>; <i>Mtssa</i>., <i>Miltassia</i>; <i>Odm</i>., <i>Odontoglossum</i>; <i>Odcdm</i>., <i>Odontocidium</i>; <i>Onc</i>., <i>Oncidium</i>; <i>P</i>., <i>Phalaenopsis</i>; , <i>Rod</i>., <i>Rodriguezia</i>; and <i>Tol</i>., <i>Tolumnia</i>.</p

Summary of gene patterns in Epidendroideae subfamily.

<p>Within each detected region, different species that share the same sequences are labeled with white circles; unique deletions are labeled as black circles. The same deletions found in different species are labeled as triangles of the same color. Black stars indicate insertions. ‘—’ indicates that no PCR product was obtained.</p

Primers for Epidendroideae genes and Oncidiinae phylogenetic analysis.

<p>Primer sequences, annealing position of the forward primer in <i>E. pusilla</i>, and the PCR amplification length are presented. The first 5 sets of primer were used for Epidendroideae analysis. The last 4 sets of primer were used for Oncidiinae phylogenetic analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034738#pone.0034738-Wu1" target="_blank">[9]</a>.</p