| 1. |
- Olsson, Mikael E., et al.
(author)
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Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L
- 2009
-
In: Phytochemistry. - : Elsevier BV. - 0031-9422 .- 1873-3700. ; 70:9, s. 1123-1128
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Journal article (peer-reviewed)abstract
- A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta 11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1 alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.
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| 2. |
- Teixeira, M.C., et al.
(author)
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Molecular cloning and expression analysis of three omega-6 desaturase genes from purslane (Portulaca oleracea L.)
- 2009
-
In: Biotechnology letters. - : Springer Science and Business Media LLC. - 0141-5492 .- 1573-6776. ; 31:7, s. 1089-1101
-
Journal article (peer-reviewed)abstract
- Two full-length cDNA clones of PoleFAD2 and one full-length cDNA clone of PoleFAD6, encoding omega-6 fatty acid desaturases, the key enzymes for the conversion of oleic into linoleic acid, were isolated from purslane (Portulaca oleracea L.) leaves and seeds. The deduced amino acid sequence of both isoforms of PoleFAD2 showed higher similarities to other microsomal omega-6 desaturases then to PoleFAD6 or other plastidial orthologues, and vice versa. Expression analysis by RT-PCR showed that all genes are expressed in all tissues of purslane tested, but higher levels of mRNA accumulation were detected in reproductive organs and cells that proliferate rapidly or store lipids. Wounding affected the levels of mRNA accumulation of both, FAD2 and FAD6 genes in purslane leaves, while chilling stress affected only FAD2 transcript level. The expression patterns observed reflect the discrete roles of these genes in membrane synthesis for cell division, thylakoid development, and lipid storage or in the biosynthetic pathway for the production of signaling molecules that influence plant development or defense.
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| 3. |
- Brodelius, Maria, et al.
(author)
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Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.
- 2002
-
In: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:14, s. 3570-3577
-
Journal article (peer-reviewed)abstract
- A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.
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| 4. |
- Komaraiah, Palle, et al.
(author)
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Growth behavior in plant cell cultures based on emissions detected by a multisensor array
- 2004
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In: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 20:4, s. 1245-1250
-
Journal article (peer-reviewed)abstract
- The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.
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| 5. |
- Lundberg, Peter, et al.
(author)
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A phosphorus-31 nuclear magnetic resonance study of elicitor-mediated metabolic changes in Catharanthus roseus suspension cultures
- 1997
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In: In vitro cellular & developmental biology. Plant. - : Springer. - 1054-5476 .- 1475-2689. ; 33:4, s. 301-305
-
Journal article (peer-reviewed)abstract
- The induction of metabolic changes in suspension cultured cells of Catharanthus roseus upon elicitation has been investigated. Addition of a yeast glucan preparation to the growth medium resulted in induction of phenylalanine ammonia lyase. Phosphate uptake and metabolism of elicited cells was followed by 31P nuclear magnetic resonance. The uptake rate of Pi from the medium by oxygenated cells of C. roseus was reduced immediately after elicitation. Despite this reduced Pi uptake elicited cells had significantly increased amounts of ATP (twofold increase within 6 h). Cytoplasmic levels of Pi, phosphomonoesters, and Uridine Diphasphate glucose (UDP-Glc) were unaffected by eliciation. Furthermore, the cytoplasmic and vacuolar pH remained constant after addition of elicitor.
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| 6. |
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| 7. |
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| 8. |
- Gliszczynska, Anna, et al.
(author)
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Sesquiterpene coumarins
- 2012
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In: Phytochemistry Reviews. - : Springer Science and Business Media LLC. - 1568-7767 .- 1572-980X. ; 11:1, s. 77-96
-
Journal article (peer-reviewed)abstract
- Plants have a long history as therapeutic tools in the treatment of human diseases and have been used as a source of medicines for ages. In search of new biologically active natural products, many plants and herbs used in traditional medicine are screened for natural products with pharmacological activity. In this paper, we present a group of natural products, the sesquiterpene coumarins isolated from plants, and describe their wide range of biological activity. Sesquiterpene coumarins are found in some plants of the families Apiaceae (Umbelliferae), Asteraceae (Compositae) and Rutaceae. The coumarin moiety is often umbelliferone (7-hydroxycoumarin) but scopo- letin (7-hydroxy-6-methoxycoumarin) and isofraxidin (7-hydroxy-6,8-dimethoxycoumarin) are also found. These coumarins are linked to a C15 terpene moiety through an ether linkage. Another group of sesquiter- pene coumarins is the prenylated 4-hydroxycoumarins where the link between the coumarin and the C15 terpene moiety is a C–C-bond at carbon 3 of the coumarin moiety. Finally, the prenylfurocoumarin-type sesquiterpenoids are a separate group of sesquiterpene coumarins based on the suggested biosynthetic pathway. Our relatively limited knowledge on the biosynthesis of sesquiterpene coumarins is reviewed.
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| 9. |
- Guo, Ming, et al.
(author)
-
A facile synthesis of molecularly imprinted polymers and their properties as electrochemical sensors for ethyl carbamate analysis
- 2018
-
In: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 8:69, s. 39721-39730
-
Journal article (peer-reviewed)abstract
- New molecularly imprinted polymers (MIPs), which exhibit specific recognition of ethyl carbamate (EC) have been synthesized and studied. In this process, EC was the template molecule and -cyclodextrin derivatives were employed as functional monomers in the molecular imprinting technique (MIT). An EC molecularly imprinted sensor (EC-MIS) was prepared by using MIT surface modification. The EC-MIS was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. EC detection performance, binding parameters and dynamics mechanism were investigated. The result showed that the synthetic route designed was appropriate and that new MIP and EC-MIS were successfully prepared. The EC-MIS exhibited a good molecular recognition of EC. A linear relationship between current and EC concentration was observed using cyclic voltammetry and the detection limit was 5.86 g L-1. The binding constant (K = 4.75 x 10(6) L mol(-1)) between EC and the EC-MIS, as well as, the number of binding sites (n = 1.48) has been determined. The EC-MIS recognition mechanism for the EC is a two-step process. The sensor was applied for the determination of EC in Chinese yellow wines, and the results were in good agreement with the gas chromatography-mass spectrometry (GC-MS) method.
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| 10. |
- Guo, Ming, et al.
(author)
-
alpha-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of alpha-Mangostin to HSA or TRF
- 2016
-
In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:9
-
Journal article (peer-reviewed)abstract
- In order to obtain the biological active compound, alpha-mangostin, from the traditional native mangosteen (Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of a-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of alpha-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (H-1 NMR and C-13 NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained alpha-mangostin was 95.6% as determined by HPLC analysis. The binding of native alpha-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that amangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to alpha-mangostin is 6.4832x10(5) L/mol and 1.4652x10(5) L/mol at 298 K and 7.8619x10(5) L/mol and 1.1582x10(5) L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between alpha-mangostin and HSA or TRF were also obtained by virtue of the Forster theory of non-radiation energy transfer. The effect of alpha-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between amangostin and HSA is hydrophobic interactions, while the main interaction between alpha-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results.
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| 11. |
- Guo, Ming, et al.
(author)
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Comparison of the interaction between lactoferrin and isomeric drugs
- 2017
-
In: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy. - : Elsevier. - 1386-1425 .- 1873-3557. ; 173, s. 593-607
-
Journal article (peer-reviewed)abstract
- The binding properties of pentacyclic triterpenoid isomeric drugs, i.e. ursolic acid (UA) and oleanolic acid (OA), to bovine lactoferrin (BLF) have been studied by molecule modeling, fluorescence spectroscopy, UV-visible absorbance spectroscopy and infrared spectroscopy (IR). Molecular docking, performed to reveal the possible binding mode or mechanism, suggested that hydrophobic interaction and hydrogen bonding play important roles to stabilize the complex. The results of spectroscopic measurements showed that the two isomeric drugs both strongly quenched the intrinsic fluorescence of BLF through a static quenching procedure although some differences between UM and OA binding strength and non-radiation energy transfer occurred within the molecules. The number of binding sites was 3.44 and 3.10 for UA and OA, respectively, and the efficiency of Forster energy transfer provided a distance of 0.77 and 1.21 nm for UA and OA, respectively. The conformation transformation of BLF affected by the drugs conformed to the "all-or-none" pattern. In addition, the changes of the ratios of alpha-helices, beta-sheets and beta-turns of BLF during the process of the interaction were obtained. The results of the experiments in combination with the calculations showed that there are two modes of pentacyclic triterpenoid binding to BLF instead of one binding mode only governed by the principle of the lowest bonding energy.
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| 12. |
- Guo, Ming, et al.
(author)
-
Synthesis, properties and applications of self-repairing carbohydrates as smart materials via thermally reversible DA bonds
- 2021
-
In: Polymers for Advanced Technologies. - : John Wiley & Sons. - 1042-7147 .- 1099-1581. ; 32:3, s. 1026-1037
-
Journal article (peer-reviewed)abstract
- Self-healing carbohydrate polymers were synthesized by Diels-Alder reaction. Intermediate products and the carbohydrate matrices were characterized by Fourier transform infrared spectroscopy (FT-IR) and H-1 NMR, while the thermally reversible properties were assessed by FT-IR and differential scanning calorimetry. The mechanical properties, water absorption, and enzymatic degradation of starch/PVA/modified carbohydrate films were examined, as well as the relationship of the properties to the DA and rDA reactions. These results showed that DA bonds were introduced into the carbohydrate polymers successfully and endow the material with self-healing thermal recyclability. The mixed films exhibited alternating strong and weak mechanical properties upon cycling through the DA and rDA reactions. Water absorption was limited and the films demonstrated good water resistance. The status of the DA bonds was found not to affect the enzymatic degradation rates of the various carbohydrate films.
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| 13. |
- Guo, Ming, et al.
(author)
-
The Difference of Serum Protein Transport between Echinosides and Verbascoside
- 2020
-
In: Journal of the Chemical Society of Pakistan. - : The chemical Society of Pakistan. - 0253-5106. ; 42:3, s. 369-382
-
Journal article (peer-reviewed)abstract
- Verbascoside (VER) is the enzymatic hydrolysis product of echinacoside (ECH). The molecular structures of ECH and VER have different glucosyl groups so they bind to serum albumin in different ways, resulting in different pharmacological actions. In this report, we have examined the binding characteristics between human serum albumin (HSA) and ECH/VER by molecular modeling and spectroscopic approaches. Molecular modeling revealed that VER bound to HSA mainly through hydrogen bonds, van der Waals forces and hydrophobic forces. The spectroscopic results showed that the interactions between HSA and VER/ECH involved a static binding process, and the bonding strength of the VER-HSA complex was stronger than that of the ECH-HSA complex. The value of the binding distances (r) was low, which indicated the occurrence of energy transfer. The reaction conformational pattern of HSA-VER and HSA-ECH gave a "two-state model" based on fluorescent phase diagram analysis. According to the thermodynamic model, the main forces between interaction of VER and HSA were hydrogen bonds and van der Waals forces, whereas the interaction between ECH and HSA was hydrophobic force. The fluorescence polarization analysis demonstrated that the interaction between HSA and VER or ECH generated a non-covalent complex. Compared with ECH, VER was more likely to bind with HSA because of its smaller molecular size and low polarity. The results of the spectral analysis concurred with the molecular modeling data, which provides a helpful reference for the study of the molecular reaction mechanism of VER/ECH binding to HSA.
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| 14. |
- Han, Junli, et al.
(author)
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Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants
- 2014
-
In: Phytochemistry. - : Elsevier BV. - 0031-9422 .- 1873-3700. ; 102, s. 89-96
-
Journal article (peer-reviewed)abstract
- The effective anti-malarial medicine artemisinin is costly because of the low content in Artemisia annua. Genetic engineering of A. annua is one of the most promising approaches to improve the yield of artemisinin. In this work, the transcription factor AaWRKY1, which is thought to be involved in the regulation of artemisinin biosynthesis, was cloned from A. annua var. Chongqing and overexpressed using the CaMV35S promoter or the trichome-specific CYP71AV1 promoter in stably transformed A. annua plants. The transcript level of AaWRKY1 was increased more than one hundred times under the CaMV35S promoter and about 40 times under the CYP71AV1 promoter. The overexpressed AaWRKY1 activated the transcription of CYP71AV1 and moreover the trichome-specific overexpression of AaWRKY1 improved the transcription of CYP71AV1 much more effectively than the constitutive overexpression of AaWRKY1, i.e. up to 33 times as compared to the wild-type plant. However the transcription levels of FDS, ADS, and DBR2 did not change significantly in transgenic plants. The significantly up-regulated CYP71AV1 promoted artemisinin biosynthesis, i.e. up to about 1.8 times as compared to the wild-type plant. It is demonstrated that trichome-specific overexpression of AaWRKY1 can significantly activate the transcription of CYP71AV1 and the up-regulated CYP71AV1 promotes artemisinin biosynthesis.
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| 15. |
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| 16. |
- Kanagarajan, Selvaraju, et al.
(author)
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Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana.
- 2012
-
In: Plant Cell Reports. - : Springer Berlin/Heidelberg. - 0721-7714 .- 1432-203X. ; 31:7, s. 1309-1319
-
Journal article (peer-reviewed)abstract
- Artemisia annua L. produces a number of sesquiterpene synthases, which catalyze the conversion of farnesyl diphosphate to various sesquiterpenes. The cDNAs encoding amorpha-4,11-diene synthase (ADS), a key enzyme in the artemisinin biosynthesis, and epi-cedrol synthase (ECS), a complex sesquiterpene cyclization syn- thase, were cloned into Cowpea mosaic virus-based viral vector (pEAQ-HT) with Kozak consensus motif and C-terminal histidine tag. The plasmids were transformed into Agrobacterium LBA4404 and, agroinfiltrated into Nicotiana benthamiana leaves along with vector (pJL3:p19) containing Tomato bushy stunt virus post- transcriptional gene silencing suppressor. Quantitative PCR was carried out to measure the transcript levels at 0, 3, 6, 9, 12 and 15 days post-infiltration (dpi). The highest relative expression was observed at 9 dpi for both genes. Transiently expressed recombinant proteins of ADS and ECS were confirmed by SDS-PAGE and western blot. Recombinant proteins were extracted from 9 dpi leaves and purified by immobilized metal ion affinity chroma- tography using histidine tag, which produced yields of 90 and 96 mg kg-1 fresh weight of leaves for ADS and ECS, respectively. Activities of the purified enzymes were assayed using gas chromatography–mass spectrometry for product identification and quantification using valencene as internal standard. The recombinant ADS and ECS con- verted farnesyl diphosphate into amorpha-4,11-diene (97 %) and epi-cedrol (96 %) as the major products, respectively. The purified enzymes exhibited the specific activity of 0.002 and 0.01 mmol min-1 mg-1 protein for ADS and ECS, respectively. The apparent kcat values were 2.1 x 10-3 s-1 and 11 x 10-3 s-1 for ADS and ECS, respectively.
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| 17. |
- Kanagarajan, Selvaraju, et al.
(author)
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Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana
- 2012
-
In: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 7:3, s. 1-10
-
Journal article (peer-reviewed)abstract
- The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6xHis) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6xHis tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.
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| 18. |
- Liu, Meng, et al.
(author)
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Characterization of a trichome-specific promoter of the aldehyde dehydrogenase 1 (ALDH1) gene in Artemisia annua
- 2016
-
In: Plant Cell Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 126:3, s. 469-480
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Journal article (peer-reviewed)abstract
- Artemisinin is a frequently used anti-malaria drug extracted from glandular trichomes (GSTs) in Artemisia annua L. In this study, we report on the characterization of the promoter of aldehyde dehydrogenase 1 (ALDH1) involved in the biosynthesis of artemisinin. A 1620-bp promoter fragment was cloned upstream of the ALDH1 start codon. Putative regulatory cis-acting elements are predicted by software, revealing that this gene is affected by complex factors. The activity of the ALDH1 promoter was analyzed using a reporter gene GUS. GUS expression showed a spatial difference in leaves at different ages. In young leaves, GUS staining was exclusively discovered in GSTs. In older leaves, both GSTs and T-shaped trichomes (TSTs) showed GUS signals. Only TSTs showed GUS staining in lower leaves. No GUS staining was detected in the bottom leaves. The result demonstrates that the ALDH1 promoter is trichome-specific. The RT-Q-PCR analysis revealed that both wild-type and recombinant promoters showed similar activity in A. annua. After application of exogenous 100 μM methyl jasmonate, 100 μM gibberellin and 100 μM salicylic acid separately, the transcript levels were increased significantly, indicating that ALDH1 may play an important role in the response to hormones in A. annua.
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| 19. |
- Matias-Hernandez, Luis, et al.
(author)
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AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana
- 2017
-
In: The Plant Journal. - : Wiley-Blackwell. - 0960-7412 .- 1365-313X. ; 90:3, s. 520-534
-
Journal article (peer-reviewed)abstract
- The effective anti-malarial drug artemisinin (AN) isolated from Artemisia annua is relatively expensive due to the low AN content in the plant as AN is only synthesized within the glandular trichomes. Therefore, genetic engineering of A. annua is one of the most promising approaches for improving the yield of AN. In this work, the AaMYB1 transcription factor has been identified and characterized. When AaMYB1 is overexpressed in A. annua, either exclusively in trichomes or in the whole plant, essential AN biosynthetic genes are also overexpressed and consequently the amount of AN is significantly increased. Artemisia AaMYB1 constitutively overexpressing plants displayed a greater number of trichomes. In order to study the role of AaMYB1 on trichome development and other possibly connected biological processes, AaMYB1 was overexpressed in Arabidopsis thaliana. To support our findings in Arabidopsis thaliana, an AaMYB1 orthologue from this model plant, AtMYB61, was identified and atmyb61 mutants characterized. Both AaMYB1 and AtMYB61 affected trichome initiation, root development and stomatal aperture in A. thaliana. Molecular analyses indicated that two crucial trichome activator genes are misexpressed in atmyb61 mutant plants and in plants overexpressing AaMYB1. Furthermore, AaMYB1 and AtMYB61 are also essential for gibberellin (GA) biosynthesis and degradation in both species by positively affecting the expression of the enzymes that convert GA(9) into the bioactive GA(4) as well as the enzymes involved in the degradation of GA(4). Overall, these results identify AaMYB1/AtMYB61 as a key component of the molecular network that connects important biosynthetic processes, and reveal its potential value for AN production through genetic engineering.
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| 20. |
- Muthusamy, Sarala Devi, et al.
(author)
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Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro
- 2020
-
In: PeerJ. - : PeerJ Inc. - 2167-8359. ; 8, s. 1-21
-
Journal article (peer-reviewed)abstract
- The sesquiterpene beta-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, beta-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of beta-caryophyllene. In this study, a full-length cDNA of a new duplicated beta-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce beta-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt-nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg(-1). fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce p-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography-mass spectrometry (GC-MS). GC-MS results revealed that the AaCPS1 produced maximum 26.5 +/- 1 mg of P-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.
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| 21. |
- Olofsson, Linda, et al.
(author)
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Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L
- 2011
-
In: BMC Plant Biology. - : BioMed Central. - 1471-2229. ; 11
-
Journal article (peer-reviewed)abstract
- Background: Recently, Artemisia annua L. (annual or sweet wormwood) has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for treatment of malaria. The plant produces relatively small amounts of artemisinin and a worldwide shortage of the drug has led to intense research in order to increase the yield of artemisinin. In order to improve our understanding of terpene metabolism in the plant and to evaluate the competition for precursors, which may influence the yield of artemisinin, we have used qPCR to estimate the expression of 14 genes of terpene metabolism in different tissues.Results: The four genes of the artemisinin biosynthetic pathway (amorpha-4,11-diene synthase, amorphadiene-12-hydroxylase, artemisinic aldehyde ∆11(13) reductase and aldehyde dehydrogenase 1) showed remarkably higher expression (between ~40- to ~500-fold) in flower buds and young leaves compared to other tissues (old leaves, stems, roots, hairy root cultures). Further, dihydroartemisinic aldehyde reductase showed a very high expression only in hairy root cultures. Germacrene A and caryophyllene synthase were mostly expressed in young leaves and flower buds while epi-cedrol synthase was highly expressed in old leaves. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase exhibited lower expression in old leaves compared to other tissues. Farnesyldiphosphate synthase, squalene synthase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase showed only modest variation in expression in the different tissues, while expression of 1-deoxy-D-xylulose-5-phosphate synthase was 7-8-fold higher in flower buds and young leaves compared to old leaves.Conclusions: Four genes of artemisinin biosynthesis were highly expressed in flower buds and young leaves (tissues showing a high density of glandular trichomes). The expression of dihydroartemisinic aldehyde reductase has been suggested to have a negative effect on artemisinin production through reduction of dihydroartemisinic aldehyde to dihydroartemisinic alcohol. However, our results show that this enzyme is expressed only at low levels in tissues producing artemisinin and consequently its effect on artemisinin production may be limited. Finally, squalene synthase but not other sesquiterpene synthases appears to be a significant competitor for farnesyl diphosphate in artemisinin-producing tissues.
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| 22. |
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| 23. |
- Olofsson, Linda, et al.
(author)
-
Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.
- 2012
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In: Plant Science. - : Elsevier BV. - 0168-9452 .- 1873-2259. ; 183, s. 9-13
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Journal article (peer-reviewed)abstract
- The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.
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| 24. |
- Prenosil, J E, et al.
(author)
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Purine Alkaloid Producing Cell Cultures: Fundamental Aspects and Possible Applications in Biotechnology
- 1987
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In: Enzyme and microbial technology. - : Elsevier BV. - 0141-0229 .- 1879-0909. ; 9:8, s. 450-458
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Journal article (peer-reviewed)abstract
- Coffea arabica is one of the plant species that has been widely studied with attention largely being given to its secondary products, caffeine and other purine alkaloids. The biosynthesis and significance of these alkaloids for the plant are elucidated and presented. Tissue cell culture and fundamental aspects of cell growth and alkaloid productivity are also discussed. The feasibility of Coffea cultivation in cell suspension has recently attracted the interest of many researchers. Although this cultivation is not of commercial interest, Coffea is especially suitable as a model cell line for reaction engineering studies because the purine alkaloids are well-characterised and readily released in culture medium. The use of free and immobilized coffee cells in various types of bioreactors (stirred tank, expanded bed, and membrane device) is shown.
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| 25. |
- Schiebe, Christian, et al.
(author)
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Inducibility of chemical defenses in Norway spruce bark is correlated with unsuccessful mass attacks by the spruce bark beetle
- 2012
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In: Oecologia. - : Springer Science and Business Media LLC. - 0029-8549 .- 1432-1939. ; 170:1, s. 183-198
-
Journal article (peer-reviewed)abstract
- Secondary attraction to aggregation pheromones plays a central role in the host colonization behavior of the European spruce bark beetle Ips typographus. However, it is largely unknown how the beetles pioneering an attack locate suitable host trees, and eventually accept or reject them. To find possible biomarkers for host choice by I. typographus, we analyzed the chemistry of 58 Norway spruce (Picea abies) trees that were subsequently either (1) successfully attacked and killed, (2) unsuccessfully attacked, or (3) left unattacked. The trees were sampled before the main beetle flight in a natural Norway spruce-dominated forest. No pheromones were used to attract beetles to the experimental trees. To test the trees' defense potential, each tree was treated in a local area with the defense hormone methyl jasmonate (MeJ), and treated and untreated bark were analyzed for 66 different compounds, including terpenes, phenolics and alkaloids. The chemistry of MeJ-treated bark correlated strongly with the success of I. typographus attack, revealing major chemical differences between killed trees and unsuccessfully attacked trees. Surviving trees produced significantly higher amounts of most of the 39 analyzed mono-, sesqui-, and diterpenes and of 4 of 20 phenolics. Alkaloids showed no clear pattern. Differences in untreated bark were less pronounced, where only 1,8-cineole and (-)-limonene were significantly higher in unsuccessfully attacked trees. Our results show that the potential of individual P. abies trees for inducing defense compounds upon I. typographus attack may partly determine tree resistance to this bark beetle by inhibiting its mass attack.
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| 26. |
- Shen, Qian, et al.
(author)
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The Genome of Artemisia annua Provides Insight into the Evolution of Asteraceae Family and Artemisinin Biosynthesis
- 2018
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In: Molecular Plant. - : Cell Press. - 1674-2052 .- 1752-9867. ; 11:6, s. 776-788
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Journal article (peer-reviewed)abstract
- Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin.
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| 27. |
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| 28. |
- Wang, Bo, et al.
(author)
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Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua
- 2016
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In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 38, s. 159-169
-
Journal article (peer-reviewed)abstract
- Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.
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| 29. |
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| 30. |
- Wang, Hongzhen, et al.
(author)
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Studies on the expression of linalool synthase using a promoter-beta-glucuronidase fusion in transgenic Artemisia annua
- 2014
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In: Journal of plant physiology (Print). - : ELSEVIER GMBH. - 0176-1617 .- 1618-1328. ; 171:2, s. 85-96
-
Journal article (peer-reviewed)abstract
- Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to beta-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652 bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that US expression will most likely have little or no effect on artemisinin production. (C) 2013 Elsevier GmbH. All rights reserved.
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| 31. |
- Wang, Hongzhen, et al.
(author)
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Studies on the Expression of Sesquiterpene Synthases Using Promoter-b-Glucuronidase Fusions in Transgenic Artemisia annua L
- 2013
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:11
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Journal article (peer-reviewed)abstract
- In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for beta-caryophyllene (CPS), epi-cedrol (ECS) and beta-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.
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| 32. |
- Wang, Hongzhen, et al.
(author)
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Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in Artemisia annua L., as Reported by a Promoter-GUS Fusion
- 2011
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In: American Journal of Plant Sciences. - : Scientific Research Publishing, Inc.. - 2158-2742 .- 2158-2750. ; 2:4, s. 619-628
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Journal article (peer-reviewed)abstract
- Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the ␣-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activ- ity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.
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| 33. |
- Wang, Hongzhen, et al.
(author)
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Trichome-specific expression of the amorpha-4,11-diene 12-hydroxylase (cyp71av1) gene, encoding a key enzyme of artemisinin biosynthesis in Artemisia annua, as reported by a promoter-GUS fusion
- 2013
-
In: Plant Molecular Biology. - : Springer Science and Business Media LLC. - 0167-4412 .- 1573-5028. ; 81:1-2, s. 119-138
-
Journal article (peer-reviewed)abstract
- Artemisinin derivatives are effective anti-malarial drugs. In order to design transgenic plants of Artemisia annua with enhanced biosynthesis of artemisinin, we are studying the promoters of genes encoding enzymes involved in artemisinin biosynthesis. A 1,151 bp promoter region of the cyp71av1 gene, encoding amorpha-4,11-diene 12-hydroxylase, was cloned. Alignment of the cloned promoter and other cyp71av1 promoter sequences indicated that the cyp71av1 promoter may be different in different A. annua varieties. Comparison to the promoter of amorpha-4,11-diene synthase gene showed a number of putative cis-acting regulatory elements in common, suggesting a co-regulation of the two genes. The cyp71av1 promoter sequence was fused to the beta-glucuronidase (GUS) reporter gene and two varieties of A. annua and Nicotiana tabacum were transformed. In A. annua, GUS expression was exclusively localized to glandular secretory trichomes (GSTs) of leaf primordia and top expanded leaves. In older leaves, there is a shift of expression to T-shaped trichomes (TSTs). Only TSTs showed GUS staining in lower leaves and there is no GUS staining in old leaves. GUS expression in flower buds was specifically localized to GSTs. The recombinant promoter carries the cis-acting regulatory elements required for GST-specific expression. The cyp71av1 promoter shows activity in young tissues. The recombinant promoter was up to 200 times more active than the wild type promoter. GUS expression in transgenic N. tabacum was localized to glandular heads. Transcript levels were up-regulated by MeJA. Wound responsiveness experiment showed that the cyp71av1 promoter does not appear to play any role in the response of A. annua to mechanical stress.
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| 34. |
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| 35. |
- Yang, Ke
(author)
-
Regulation of artemisinin biosynthesis in Artemisia annua L
- 2015
-
Doctoral thesis (other academic/artistic)abstract
- Artemisinin and its derivatives are the most powerful medicines against malaria. However,the low annual yield of artemisinin from industry restricts the usage of this effective drug inendemic areas such as Africa. Nowadays the artemisinin industry mainly depends onextractions from Artemisia annua L. plants, which unfortunately accumulate very littleartemisinin for most varieties. In order to increase artemisinin content in these plants, wemust understand deeply and clearly how the biosynthetic pathway of artemisinin in plantsworks. Many key enzymes have been found and we need to study the genes that encodethem, as well as how these genes are regulated.Amorpha-4,11-diene 12-hydroxylase (CYP71AV1) is one of the key enzymes of theartemisinin biosynthetic pathway. This gene is specifically expressed in glandular trichomes,where artemisinin is synthesized and stored. We cloned the promoter of this gene and withthe GUS fusion assay we managed to confirm that the promoter of this gene is trichomespecific. A few putative cis-acting regulatory elements may control the promoter activity.Artemisinic aldehyde 11(13) reductase (DBR2) is also an important enzyme of thepathway. It allows one of the precursors of artemisinin, i.e. artemisinic aldehyde, to gotowards artemisinin rather than arteannuin B. The gene expression level also differsbetween high artemisinin producer varieties and low artemisinin producer varieties. Theformer group expresses DBR2 much more than the latter group. According to our results,the promoter of the DBR2 gene appears to be the key reason for the different expressionlevel of DBR2 in high and low producers.How the artemisinin biosynthetic pathway is regulated has not been revealed to any greatextent. We used wounding treatment on A. annua plants and found that when the plantsresponded to wounding stress, their artemisinin content increased. Some key genes of thepathway also showed an increased expression. This suggests that artemisinin may be relatedto wounding stress responses and that the regulation factors of the pathway are upregulatedupon wounding stress.The most common regulation factors for plant secondary metabolites biosynthesis aretranscription factors. We cloned an AaMYB1 gene from the MYB family. The geneticallymodified plants, which over-expressed AaMYB1 were tested for their artemisinin content,as well as the expression level of some key genes of the pathway. The artemisinin contentincreased in these transgenic plants and the key genes were also up-regulated by AaMYB1.In addition, over-expressing AaMYB1 in Arbidopsis thaliana shows that the gene is able toregulate trichome initiation and development as well as gibberellic acid biosynthesis, whichis related to early flowering.
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| 36. |
- Yang, Ke, et al.
(author)
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The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.
- 2015
-
In: Plant Molecular Biology. - : Springer Science and Business Media LLC. - 0167-4412 .- 1573-5028. ; 88:4-5, s. 325-340
-
Journal article (peer-reviewed)abstract
- The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the ‘2/39’, ‘Chongqing’ and ‘Anamed’ varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the ‘Meise’, ‘Iran#8’, ‘Iran#14’, ‘Iran#24’ and ‘Iran#47’ varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties. © 2015, Springer Science+Business Media Dordrecht.
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