AaABF3, an Abscisic Acid–Responsive Transcription Factor, Positively Regulates Artemisinin Biosynthesis in Artemisia annua

Artemisinin is well known for its irreplaceable curative effect on the devastating parasitic disease, Malaria. This sesquiterpenoid is specifically produced in Chinese traditional herbal plant Artemisia annua. Earlier studies have shown that phytohormone abscisic acid (ABA) plays an important role in increasing the artemisinin content, but how ABA regulates artemisinin biosynthesis is still poorly understood. In this study, we identified that AaABF3 encoded an ABRE (ABA-responsive elements) binding factor. qRT-PCR analysis showed that AaABF3 was induced by ABA and expressed much higher in trichomes where artemisinin is synthesized and accumulated. To further investigate the mechanism of AaABF3 regulating the artemisinin biosynthesis, we carried out dual-luciferase analysis, yeast one-hybrid assay and electrophoretic mobility shift assay. The results revealed that AaABF3 could directly bind to the promoter of ALDH1 gene, which is a key gene in artemisinin biosynthesis, and activate the expression of ALDH1. Functional analysis revealed that overexpression of AaABF3 in A. annua enhanced the production of artemisinin, while RNA interference of AaABF3 resulted in decreased artemisinin content. Taken together, our results demonstrated that AaABF3 played an important role in ABA-regulated artemisinin biosynthesis through direct regulation of artemisinin biosynthesis gene, ALDH1

An Investigation on the Applicability of the Integrated Method for Multi-Carrier Energy Flow Analysis

The integrated energy system (IES) combined with electricity and heat networks is a research hotspot in energy field in recent years. As the foundation of security and economy analysis of IES, the steady-state multi-carrier energy flow (MEF) analysis is important but difficult to converge. An integrated method for solving MEF has been presented and adopted in some published literatures. In this paper, we investigate the integrated method and point out its two problems: one is the failure of the integrated method when energy reverses during iteration in some exceptional cases; the other is the high sensitivity of the method to the initial value. Thereafter, the fundamental causes for the two problems are revealed. Moreover, we modify the integrated method based on the fundamental causes to deal with the two problems. The cost and benefit of the modified integrated method are analyzed as well. Finally, the integrated method and the modified integrated method are discussed on the basis of theoretical and simulation results. The results show that with fewer iteration times and less calculation time, the modified integrated method has better applicability to exceptional cases and less sensitivity to the initial value

Roles of MPBQ-MT in Promoting α/γ-Tocopherol Production and Photosynthesis under High Light in Lettuce

<div><p>2-methyl-6-phytyl-1, 4-benzoquinol methyltransferase (MPBQ-MT) is a vital enzyme catalyzing a key methylation step in both α/γ-tocopherol and plastoquinone biosynthetic pathway. In this study, the gene encoding MPBQ-MT was isolated from lettuce (<i>Lactuca sativa</i>) by rapid amplification of cDNA ends (RACE), named <i>LsMT</i>. Overexpression of <i>LsMT</i> in lettuce brought about a significant increase of α- and γ-tocopherol contents with a reduction of phylloquinone (vitamin K1) content, suggesting a competition for a common substrate phytyl diphosphate (PDP) between the two biosynthetic pathways. Besides, overexpression of <i>LsMT</i> significantly increased plastoquinone (PQ) level. The increase of tocopherol and plastoquinone levels by <i>LsMT</i> overexpression conduced to the improvement of plants’ tolerance and photosynthesis under high light stress, by directing excessive light energy toward photosynthetic production rather than toward generation of more photooxidative damage. These findings suggest that the role and function of <i>MPBQ-MT</i> can be further explored for enhancing vitamin E value, strengthening photosynthesis and phototolerance under high light in plants.</p></div

Expression levels of genes encoding HPPD, HPT, MPBQ-MT, TC and γ-TMT involved in tocopherol biosynthesis in wild type (WT) and transgenic lines (M1-M4), measured by quantitative real-time PCR.

<p><i>Ubiquitin</i> was used as a control for normalization. mRNA expression levels (y-axis) of the five genes were measured with 2^-ΔCt. Data is the mean value ± SD of four biological replicates from T1 progenies. Asterisk indicated a significant difference compared to the value of WT (Student’s <i>t</i>-test, P<0.05).</p

Measurements of chlorophyll fluorescence, photooxidation products, NPQ level and soluble sugar content in wild type (WT) and transgenic plants’ leaves under high light condition (photon flux density: 1000μmol/m²/s, 16h light/8h dark) for 6 days.

<p>The measurements were taken after 0, 3, 6 days of the treatment. (A) the actual quantum yield of PSⅡ-mediated electron transport, (B) the maximum quantum yield of PSⅡ photochemistry, (C) H₂O₂ content, (D) malondialdehyde (MDA) content, (E) NPQ level and (F) soluble sugar content. Data is the mean value ± SD of four biological replicates from T1 progenies. Asterisk indicated a significant difference compared to the value of WT (Student’s <i>t</i>-test, P<0.05).</p

Biosynthetic pathways of vitamin E, vitamin K1, carotenoid and plastoquinone.

<p>Substrate abbreviations: DHNA, 1,4-dihydroxy-2-naphtoate; DMPBQ, 2,3-dimethyl-5-phytyl benzoquinol; DMPQ, demethylphylloquinone; DMAPP, dimethylallyl diphosphate; GGDP, geranylgeranyl diphosphate; HGA, homogentisate; HPP, ρ-hydroxyphenylpyruvate; IPP, isopentenyl diphosphate; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinol; MSBQ, 2-methyl-6-solanesyl-1,4-benzoquinol; PDP, phytyl diphosphate; SDP, solanesyl diphosphate. Enzyme abbreviations: DHNA-PT, DHNA phytyl transferase; DMPQ-MT, DMPQ methyltransferase; GGPS, geranylgeranyl diphosphate synthase; GGR, geranylgeranyl reductase; HPPD, HPP dioxygenase; HPT, homogentisate phytyl transferase; HST, homogentisate solanesyl transferase; MPBQ/MSBQ-MT, MPBQ/MSBQ methyltransferase; PSY, phytoene synthase; TC, tocopherol cyclase; γ-TMT, γ-tocopherol methyltransferase.</p

Relative plastoquinone level in wild type (WT) and transgenic lines (M1-M4).

<p>Data is the mean value ± SD of four biological replicates from T1 progenies. Asterisk indicated a significant difference compared to the value of WT (Student’s <i>t</i>-test, P<0.05).</p