Artemisinin is a sesquiterpene lactone produced by the Chinese traditional herb Artemisia annua and is used for the treatment of malaria. It is known that salicylic acid (SA) can enhance artemisinin ...content but the mechanism by which it does so is not known. In this study, we systematically investigated a basic leucine zipper family transcription factor, AaTGA6, involved in SA signaling to regulate artemisinin biosynthesis. We found specific in vivo and in vitro binding of the AaTGA6 protein to a 'TGACG' element in the AaERF1 promoter. Moreover, we demonstrated that AaNPR1 can interact with AaTGA6 and enhance its DNA-binding activity to its cognate promoter element 'TGACG' in the promoter of AaERF1, thus enhancing artemisinin biosynthesis. The artemisinin contents in AaTGA6-overexpressing and RNAi transgenic plants were increased by 90-120% and decreased by 20-60%, respectively, indicating that AaTGA6 plays a positive role in artemisinin biosynthesis. Importantly, heterodimerization with AaTGA3 significantly inhibits the DNA-binding activity of AaTGA6 and plays a negative role in target gene activation. In conclusion, we demonstrate that binding of AaTGA6 to the promoter of the artemisinin-regulatory gene AaERF1 is enhanced by AaNPR1 and inhibited by AaTGA3. Based on these findings, AaTGA6 has potential value in the genetic engineering of artemisinin production.
Glandular secreting trichomes (GSTs) synthesize and secrete large quantities of secondary metabolites, some of which have well-established commercial value. An example is the anti-malarial compound ...artemisinin, which is synthesized in the GSTs of Artemisia annua. Accordingly, there is considerable interest in understanding the processes that regulate GST density as a strategy to increase artemisinin production. In this study, we identified a GST-specific WRKY transcription factor from A. annua, AaGSW2, which is positively regulated by the direct binding of the homeodomain proteins AaHD1 and AaHD8 to the L1-box of the AaGSW2 promoter. Overexpression of AaGSW2 in A. annua significantly increased GST density, while AaGSW2 knockdown lines showed impaired GST initiation. Ectopic expression of AaGSW2 homologs from two mint cultivars, Mentha spicata and Mentha haplocalyx, in A. annua also induced GST formation. These results reveal a molecular mechanism involving homeodomain and WRKY proteins that controls glandular trichome initiation, at least part of which is shared by A. annua and mint.
There are many biosynthetic pathways competing for the metabolic flux with the artemisinin biosynthetic pathway in Artemisia annua L. To study the relationship between genes encoding enzymes at ...branching points and the artemisinin biosynthetic pathway, β-caryophyllene, β-farnesene and squalene were sprayed on young seedlings of A. annua. Transient expression assays indicated that the transcription levels of β-caryophyllene synthase (CPS), β-farnesene synthase (BFS) and squalene synthase (SQS) were inhibited by β-caryophyllene, β-farnesene and squalene, respectively, while expression of some artemisinin biosynthetic pathway genes increased. Thus, inhibition of these genes encoding enzymes at branching points may be helpful to improve the artemisinin content. For further study, the expression levels of four branch pathway genes CPS, BFS, germacrene A synthase (GAS) and SQS were down-regulated by the antisense method in A. annua. In anti-CPS transgenic plants, mRNA levels of BFS and ADS were increased, and the contents of β-farnesene, artemisinin and dihydroartemisinic acid (DHAA) were increased by 212, 77 and 132%, respectively. The expression levels of CPS, SQS, GAS, amorpha-4,11-diene synthase (ADS), amorphadiene 12-hydroxylase (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1) were increased in anti-BFS transgenic plants and, at the same time, the contents of artemisinin and DHAA were increased by 77% and 54%, respectively, and the content of squalene was increased by 235%. In anti-GAS transgenic plants, mRNA levels of CPS, BFS, ADS and ALDH1 were increased. The contents of artemisinin and DHAA were enhanced by 103% and 130%, respectively. In anti-SQS transgenic plants, the transcription levels of BFS, GAS, CPS, ADS, CYP71AV1 and ALDH1 were all increased. Contents of artemisinin and DHAA were enhanced by 71% and 223%, respectively, while β-farnesene was raised to 123%. The mRNA level of artemisinic aldehyde Δ11(13) reductase (DBR2) had changed little in almost all transgenic plants.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
This research investigated heat transfer performance and flow characteristics
of three polydimethylsiloxane microchannels full of deionised water as a
working fluid. A single micropillar, horizontal ...micropillars, and vertical
micropillars along the flow direction were prepared on the microchannels
experimentally. Results show that the Nusselt number of microchannels with
two horizontal micropillars is 19% higher than that with a single
micropillar. The microchannel with two vertical micropillars has the Nusselt
number is 29% higher than that with a single micropillar, which shows the
best performance on the heat transfer enhancement. Visualization experiments
of the flow field were carried out to explore the enhanced mechanism of the
heat transfer for microchannels with various micropillar arrangements. When
the flow rate is 7 mLpm, the maximum velocities near the single cylinder
and the horizontal micro-column are 0.5 m/s and 0.52 m/s. Fluid velocity in
a region between two vertical micropillars reaches 0.72 m/s when the flow
rate is 7 mLpm. The fluid in the high-speed region is fully mixed around the
micropillar, which reduces the stagnation region area down-stream of the
vertical micropillar and enhances heat transfer.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on ...the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1
G411S
mutation that diminishes Ub kinase activity, we found that the PINK1
G411A
variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development.
Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
•Southern-blot analyses showed that ACR1, ACR10, ACR31 and ACR52 had low copies of the integration transgenes.•RT-Q-PCR showed that the expression levels of the three genes were significantly ...increased in transgenic A. annua plants.•HPLC showed that artemisinin content of ACR16 was found to contain 2.4-fold higher (15.1mg/g DW) than the control.•Overexpression of those three genes promoted the metabolic flux flows toward biosynthesis of artemisinin.
Artemisinin is an effective anti-malarial drug isolated from A. annua, which has an enormous commercialization demand all over the world. However, the low artemisinin content of A. annua greatly limits the commercialization of artemisinin. In this study, we report the results of our experiments, where for the first time we have achieved the overexpression of ADS, CYP71AV1 and CPR genes in A. annua. Eight transgenic A. annua plants were obtained through Agrobacterium tumefaciens-mediated transformation, which was confirmed by PCR. Southern-blot analyses showed that some of the transgenic lines had low copies of the integration transgenes. The results of real time-qPCR showed that the expression levels of ADS, CYP71AV1 and CPR genes were significantly increased, too. The HPLC analyses showed that the artemisinin contents were significantly increased in these transgenic plants. One of the transgenic plants, ACR16, was found to contain 2.4-fold higher (15.1mg/g DW) artemisinin than the control plants (pCAMBIA2300 transgenic plants). All above results showed that overexpression of ADS, CYP71AV1 and CPR genes in A. annua could promoted the metabolic flux flows toward biosynthesis of artemisinin and effectively increase the level of artemisinin content in transgenic A. annua plants.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Glandular trichomes and cuticles are both specialized structures that cover the epidermis of aerial plant organs. The former are commonly regarded as ‘biofactories’ for producing valuable natural ...products. The latter are generally considered as natural barriers for defending plants against abiotic and biotic stresses. However, the regulatory network for their formation and relationship remains largely elusive.
Here we identify a homeodomain-leucine zipper (HD-ZIP) IV transcription factor, AaHD8, directly promoting the expression of AaHD1 for glandular trichome initiation in Artemisia annua.
We found that AaHD8 positively regulated leaf cuticle development in A. annua via controlling the expression of cuticle-related enzyme genes. Furthermore, AaHD8 interacted with a MIXTA-like protein AaMIXTA1, a positive regulator of trichome initiation and cuticle development, forming a regulatory complex and leading to enhanced transcriptional activity in regulating the expression of AaHD1 and cuticle development genes.
Our results reveal a molecular mechanism by which a novel HD-ZIP IV/MIXTA complex plays a significant role in regulating epidermal development, including glandular trichome initiation and cuticle formation.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
Summary
The plant
Artemisia annua
is well known due to the production of artemisinin, a sesquiterpene lactone that is widely used in malaria treatment. Phytohormones play important roles in plant ...secondary metabolism, such as jasmonic acid (
JA
), which can induce artemisinin biosynthesis in
A. annua
. Nevertheless, the
JA
‐inducing mechanism remains poorly understood.
The expression of gene
Aa
MYC
2
was rapidly induced by
JA
and Aa
MYC
2 binds the G‐box‐like motifs within the promoters of gene
CYP
71
AV
1
and
DBR
2
, which are key structural genes in the artemisinin biosynthetic pathway.
Overexpression of
Aa
MYC
2
in
A. annua
significantly activated the transcript levels of
CYP
71
AV
1
and
DBR
2
, which resulted in an increased artemisinin content. By contrast, artemisinin content was reduced in the
RNA
i transgenic
A. annua
plants in which the expression of
Aa
MYC
2
was suppressed. Meanwhile, the
RNA
i transgenic
A. annua
plants showed lower sensitivity to methyl jasmonate treatment than the wild‐type plants.
These results demonstrate that Aa
MYC
2 is a positive regulator of artemisinin biosynthesis and is of great value in genetic engineering of
A. annua
for increased artemisinin production.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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