Roots Of Artemisia Annua Research Articles

Heterogeneous nutrient supply modulates root exudation and accumulation of medicinally valuable compounds in Artemisia annua and Hypericum perforatum.

Plants have evolved complex mechanisms to adapt to nutrient-deficient environments, including stimulating lateral root proliferation into local soil patches with high nutrient content in response to heterogeneous nutrient distribution. Despite the widespread occurrence of this phenomenon in soil, the effect of heterogeneous nutrient distribution on the accumulation of secondary compounds in plant biomass and their exudation by roots remains largely unknown. This study aims to fill this critical knowledge gap by investigating how deficiency and unequal distributions of nitrogen (N), phosphorus (P), and iron (Fe) affect plant growth and accumulation of the antimalarial drug artemisinin (AN) in leaves and roots of Artemisia annua, as well as AN exudation by roots. Heterogeneous N and P supplies strongly increased root exudation of AN in half of a split-root system exposed to nutrient deficiency. By contrast, exposure to a homogeneous nitrate and phosphate deficiency did not modulate root exudation of AN. This indicates that a combination of local and systemic signals, reflecting low and high nutritional statuses, respectively, were required to enhance AN exudation. This exudation response was independent of the regulation of root hair formation, which was predominantly modulated by the local signal. In contrast to the heterogeneous supply of N and P, heterogeneous Fe supply did not modulate AN root exudation but increased AN accumulation in locally Fe-deficient roots. No modulation of nutrient supply significantly changed the accumulation of AN in A. annua leaves. The impact of a heterogeneous nitrate supply on growth and phytochemical composition was also investigated in Hypericum perforatum plants. Unlike in A. annue, the uneven N supply did not significantly influence the exudation of secondary compounds in the roots of H. perforatum. However, it did enhance the accumulation of several biologically active compounds, such as hypericin, catechin, and rutin isomers, in the leaves of H. perforatum. We propose that the capacity of plants to induce the accumulation and/or differential exudation of secondary compounds under heterogeneous nutrient supply is both species- and compound-specific. The ability to differentially exude AN may contribute to A. annua's adaptation to nutrient disturbances and modulate allelopathic and symbiotic interactions in the rhizosphere.

Symbiotic fungi in roots of Artemisia annua with special reference to endophytic colonizers

Advances on plant–fungal interactions reveal that root symbiotic fungi actively modulate host growth, resistance response and secondary metabolism. Artemisia annua has been widely recognized as an important medicinal plant for artemisinin production, yet little is known about the fungal consortium associated with roots of A. annua. In this article, microscopic and culture-dependant methods were used to evaluate the identity and taxonomic affinities of root symbiotic fungi. Morphological evidence confirmed that arbuscular mycorrhizal fungi were dominant fungal group in naturally regenerated roots, but low colonization frequency in planted roots. Dark septate endophytes (DSEs) were easily found, which were characterized with dark pigmented hypha and a sclerotium-like structure in root cortex, and other endophytic fungi also occurred. A total of 36 isolates were recovered. Combined morphological and molecular identification (based on ITS sequences) determined 21 fungal taxa (genotype), which were placed into numerous lineages of Ascomycota. The best BLAST match indicated that almost half of total taxa were closely related to undescribed fungi, some of them may act as novel DSEs but experimental data were warranted. Interestingly, remarkable difference of fungal community associated with two types of roots was examined and no culturable fungi overlapped. Our findings provide some additional evidence that DSEs and other root endophytes may be as common as mycorrhizal fungi. Recovered fungi as raw materials for bioassay of endophytes-mediated promotion of artemisinin content in A. annua will be conducted in further research.

Phytomonospora endophytica gen. nov., sp. nov., isolated from the roots of Artemisia annua L.

A novel endophytic actinomycete, strain YIM 65646(T), was isolated from the roots of Artemisia annua L. collected from Yunnan Province, south-west China. Growth was observed in the temperature range 10-40 °C (optimum 20-28 °C) and at pH 6.0-9.0 (optimum pH 7.0). The organism formed well-developed, branched substrate mycelia, but aerial mycelium was not produced. Phenotypic characterization and 16S rRNA gene sequence analysis indicated that strain YIM 65646(T) belonged to the family Micromonosporaceae (sharing ≤93.6% sequence similarity with members of this family) and formed a distinct clade in the Micromonosporaceae phylogenetic tree. The strain contained meso-diaminopimelic acid in the cell wall and mannose, ribose, galactose and glucose in whole-cell hydrolysates. The major menaquinones were MK-10(H(4)), MK-10(H(2)), MK-8(H(2)), MK-9(H(2)) and MK-10(H(6)). The major fatty acids were iso-C(15:0), anteiso-C(15:0), anteiso-C(17:0), iso-C(17:0) and iso-C(16:0). The DNA G+C content of strain YIM 65646(T) was 70.0 mol%. On the basis of morphological and chemotaxonomic properties and phylogenetic analysis based on 16S rRNA gene sequence data, it is proposed that this strain should be classified in a novel genus and species, Phytomonospora endophytica gen. nov., sp. nov., in the family Micromonosporaceae. The type strain is YIM 65646(T) (=CCTCC AA 209041(T) =DSM 45386(T)).

Transformed roots of Artemisia annua exhibit an unusual pattern of border cell release

Border cells from Artemisia annua were examined from hairy roots grown in shake flasks, culture plates, a bubble column reactor, and a nutrient mist (aeroponic) reactor. When well-hydrated roots were subjected to shear, border cells were first released as an agglomerate and did not disperse for several hours. Staining with neutral red and fluorescein diacetate (FDA) showed that both agglomerates and dispersed cells were alive. It was determined that FDA is cleaved by pectin methylesterase (PME) and that PME may not be particularly active in the released agglomerates until the border cells disperse. Untransformed roots isolated from A. annua plants showed no border cell agglomerate formation and border cells readily dispersed. These results suggest that our hairy root clone is deficient in border cell release perhaps resulting from the transformation process.

Stimulation of artemisinin production in Artemisia annua hairy roots by the elicitor from the endophytic Colletotrichum sp.

Artemisinin content in hairy roots of Artemisia annua was increased from 0.8 mg g−1 dry wt to 1 mg g−1 dry wt by using elicitor treatment of mycelial extracts from the endophytic fungus Colletotrichum sp. The increase of artemisinin was dependent on the growth stage of hairy roots as well as on the dose of the elicitor applied. When hairy roots of 23-day-old cultures (later growth phase) were exposed to the elicitor at 0.4 mg total sugar ml−1 for 4 days, the maximum production of artemisinin reached to 13 mg l−1, a 44% increase over the control. This is the first report on the stimulation of artemisinin production in hairy roots by the elicitor from an endophytic fungus of A. annua.

The growth of single roots of Artemisia annua in nutrient mist reactors.

To better characterize the development and growth of hairy roots in a mist-fed root bed, a single root aerosol reactor was developed. Growth kinetics studies were conducted on hairy roots of Artemisia annua as a function of the mist cycle, carrier gas, and nutrient compositions. Sustained rapid growth was only observed when conditioned medium was fed to the roots. The presence of 1% CO(2) in the carrier gas did not enhance the growth kinetics but it did prevent necrosis of the tissue at the highest mist cycle.

Cryopreservation of Transformed (Hairy) Roots ofArtemisia annua

The antimalarial drug artemisinin has been found in transformed (hairy) roots ofArtemisia annua.A protocol was developed to preserveA. annuahairy roots in liquid nitrogen. Root tips were excised from 7-day-old cultures and held on solid White's medium for 24 h prior to cryoprotection. They were then treated with a cryoprotecting mixture containing 8% (v/v) dimethyl sulfoxide (Me2SO) and 20% (w/v) sucrose at 25°C for 1 h followed by cooling at 0.09°C/min to 4°C then cooling to −35°C at 0.72°C/min. Vials containing the root tips were then plunged into liquid nitrogen. After thawing in a water bath to 37°C, root tips were held in the cryoprotecting mixture for 10 min before it was diluted to 25% of its original concentration. Root tips were washed once with fresh liquid White's medium and held for 1 h prior to culturing on White's medium with 0.2% Gelrite and 3% (w/v) sucrose. Regrowth of root tips averaged 65%. Independent variables in this study included 1) Me2SO concentration; 2) the type and concentration of cosolutes; 3) cooling rate(s); 4) the temperature at which the sample is transferred to liquid nitrogen; 5) the age of the culture from which root tips are taken; 6) the recovery period between root tip excision and immersion in cryoprotectant; and 7) the amount of Gelrite used in the postthaw plating medium.