Melatonin imparts tolerance to combined drought and high-temperature stresses in tomato through osmotic adjustment and ABA accumulation.

In recent years, environmental stresses viz., drought and high-temperature negatively impacts the tomato growth, yield and quality. The effects of combined drought and high-temperature (HT) stresses during the flowering stage were investigated. The main objective was to assess the effects of foliar spray of melatonin under both individual and combined drought and HT stresses at the flowering stage. Drought stress was imposed by withholding irrigation, whereas HT stress was imposed by exposing the plants to an ambient temperature (AT)+5°C temperature. The drought+HT stress was imposed by exposing the plants to drought first, followed by exposure to AT+5°C temperature. The duration of individual and combined drought or HT stress was 10 days. The results showed that drought+HT stress had a significant negative effect compared with individual drought or HT stress alone. However, spraying 100 µM melatonin on the plants challenged with individual or combined drought and HT stress showed a significant increase in total chlorophyll content [drought: 16%, HT: 14%, and drought+HT: 11%], Fv/Fm [drought: 16%, HT: 15%, and drought+HT: 13%], relative water content [drought: 10%, HT: 2%, and drought+HT: 8%], and proline [drought: 26%, HT: 17%, and drought+HT: 14%] compared with their respective stress control. Additionally, melatonin positively influenced the stomatal and trichome characteristics compared with stress control plants. Also, the osmotic adjustment was found to be significantly increased in the melatonin-sprayed plants, which, in turn, resulted in an increased number of fruits, fruit set percentage, and fruit yield. Moreover, melatonin spray also enhanced the quality of fruits through increased lycopene content, carotenoid content, titratable acidity, and ascorbic acid content, compared with the stress control. Overall, this study highlights the usefulness of melatonin in effectively mitigating the negative effects of drought, HT, and drought+HT stress, thus leading to an increased drought and HT stress tolerance in tomato.

Plant Metabolomics: Current Initiatives and Future Prospects.

Plant metabolomics is a rapidly advancing field of plant sciences and systems biology. It involves comprehensive analyses of small molecules (metabolites) in plant tissues and cells. These metabolites include a wide range of compounds, such as sugars, amino acids, organic acids, secondary metabolites (e.g., alkaloids and flavonoids), lipids, and more. Metabolomics allows an understanding of the functional roles of specific metabolites in plants' physiology, development, and responses to biotic and abiotic stresses. It can lead to the identification of metabolites linked with specific traits or functions. Plant metabolic networks and pathways can be better understood with the help of metabolomics. Researchers can determine how plants react to environmental cues or genetic modifications by examining how metabolite profiles change under various crop stages. Metabolomics plays a major role in crop improvement and biotechnology. Integrating metabolomics data with other omics data (genomics, transcriptomics, and proteomics) provides a more comprehensive perspective of plant biology. This systems biology approach enables researchers to understand the complex interactions within organisms.

Genetic Diversity and Population Structure in Ethiopian Mustard (Brassica carinata A. Braun) as Revealed by Single Nucleotide Polymorphism Markers.

Ethiopian mustard (Brassica carinata A. Braun) is currently one of the potential oilseeds dedicated to the production for biofuel and other bio-industrial applications. The crop is assumed to be native to Ethiopia where a number of diversified B. carinata germplasms are found and conserved ex situ. However, there is very limited information on the genetic diversity and population structure of the species. This study aimed to investigate the genetic diversity and population structure of B. carinata genotypes of different origins using high-throughput single nucleotide polymorphism (SNP) markers. We used Brassica 90K Illumina InfiniumTM SNP array for genotyping 90 B. carinata genotypes, and a total of 11,499 informative SNP markers were used for investigating the population structure and genetic diversity. The structure analysis, principal coordinate analysis (PcoA) and neighbor-joining tree analysis clustered the 90 B. carinata genotypes into two distinct subpopulations (Pop1 and Pop2). The majority of accessions (65%) were clustered in Pop1, mainly obtained from Oromia and South West Ethiopian People (SWEP) regions. Pop2 constituted dominantly of breeding lines and varieties, implying target selection contributed to the formation of distinct populations. Analysis of molecular variance (AMOVA) revealed a higher genetic variation (93%) within populations than between populations (7%), with low genetic differentiation (PhiPT = 0.07) and poor correlation between genetic and geographical distance (R = 0.02). This implies the presence of gene flow (Nm > 1) and weak geographical structure of accessions. Genetic diversity indices showed the presence of moderate genetic diversity in B. carinata populations with an average genetic diversity value (HE = 0.31) and polymorphism information content (PIC = 0.26). The findings of this study provide important and relevant information for future breeding and conservation efforts of B. carinata.

Nanopriming Action of Microwave-Assisted Biofunctionalized ZnO Nanoparticles to Enhance the Growth under Moisture Stress in Vigna radiata.

Bare and stabilized zinc oxide nanoparticles (ZnO NPs) were prepared by a microwave-assisted method and used as a priming agent to improve the morphological, physiological, and biochemical quality of Vigna radiata. The priming action was made under normal and moisture stress conditions. A microwave reactor of 850 watts power was used to heat 30 mL of a nanocolloidal solution at 140 °C for 20 min. The stable spherical ZnO NPs at 50.4 mV with 28.2 nm particle size were generated and capped with different biomolecules, cysteine and PVA, to get biostabilized ZnO NPs at 48.8 and 108.5 nm with ζ potentials of -56.2 and -52.0 mV, respectively, holding distinct morphology. The nanopriming effect was studied in V. radiata seeds for bare ZnO and capped ZnO NPs under normal and moisture stress environments. Cysteine-capped ZnO NPs at 250 ppm showed improved germination (90 and 76%), radicle growth (7.6 and 3.6 cm), seedling Vigor (3064 and 1816), dry matter production (145.06 and 96.92 mg/25 seedlings), and hydrolytic (α-amylase and protease) and antioxidant (peroxidase and superoxide dismutase) enzyme activity under normal and moisture stress conditions. The improved priming action of cysteine-capped ZnO NPs is due to increased cell elongation and cell division in the radicle. The uptake and translocation of ZnO NPs in the V. radiata root are evidenced by the presence of an 11.4 ppm zinc level, which was also supported by EDAX and FITC labeling results.

Melatonin Enhances the Photosynthesis and Antioxidant Enzyme Activities of Mung Bean under Drought and High-Temperature Stress Conditions.

Mung bean, a legume, is sensitive to abiotic stresses at different growth stages, and its yield potential is affected by drought and high-temperature stress at the sensitive stage. Melatonin is a multifunctional hormone that plays a vital role in plant stress defense mechanisms. This study aimed to evaluate the efficiency of melatonin under individual and combined drought and high-temperature stress in mung bean. An experiment was laid out with five treatments, including an exogenous application of 100 µM melatonin as a seed treatment, foliar spray, and a combination of both seed treatment and foliar spray, as well as absolute control (ambient condition) and control (stress without melatonin treatment). Stresses were imposed during the mung bean's reproductive stage (31-40 DAS) for ten days. Results revealed that drought and high-temperature stress significantly decreased chlorophyll index, Fv/Fm ratio, photosynthetic rate, stomatal conductance, and transpiration rate through increased reactive oxygen species (ROS) production. Foliar application of melatonin at 100 µM concentration enhanced the activity of antioxidant enzymes such as superoxide dismutase, catalase, and ascorbate peroxidase and the concentration of metabolites involved in osmoregulation and ion homeostasis; thereby, it improves physiological and yield-related traits in mung bean under individual and combined stress at the reproductive stage.

Corrigendum: Improved fatty acid composition of field cress (Lepidium campestre) by CRISPR/Cas9-mediated genome editing.

[This corrects the article DOI: 10.3389/fpls.2023.1076704.].

Corrigendum: Efficient protoplast regeneration protocol and CRISPR/Cas9-mediated editing of glucosinolate transporter (GTR) genes in rapeseed (Brassica napus L.).

[This corrects the article DOI: 10.3389/fpls.2021.680859.].

Improved fatty acid composition of field cress (Lepidium campestre) by CRISPR/Cas9-mediated genome editing.

The wild species field cress (Lepidium campestre) has the potential to become a novel cover and oilseed crop for the Nordic climate. Its seed oil is however currently unsuitable for most food, feed, and industrial applications, due to the high contents of polyunsaturated fatty acids (PUFAs) and erucic acid (C22:1). As the biosynthesis of these undesirable fatty acids is controlled by a few well-known major dominant genes, knockout of these genes using CRISPR/Cas9 would thus be more effective in improving the seed oil quality. In order to increase the level of the desirable oleic acid (C18:1), and reduce the contents of PUFAs and C22:1, we targeted three important genes FATTY ACID ELONGASE1 (FAE1), FATTY ACID DESATURASE2 (FAD2), and REDUCED OLEATE DESATURASE1 (ROD1) using a protoplast-based CRISPR/Cas9 gene knockout system. By knocking out FAE1, we obtained a mutated line with almost no C22:1, but an increase in C18:1 to 30% compared with 13% in the wild type. Knocking out ROD1 resulted in an increase of C18:1 to 23%, and a moderate, but significant, reduction of PUFAs. Knockout of FAD2, in combination with heterozygous FAE1fae1 genotype, resulted in mutated lines with up to 66% C18:1, very low contents of PUFAs, and a significant reduction of C22:1. Our results clearly show the potential of CRISPR/Cas9 for rapid trait improvement of field cress which would speed up its domestication process. The mutated lines produced in this study can be used for further breeding to develop field cress into a viable crop.

Comparative Metabolomic Profiling of Horse Gram (Macrotyloma uniflorum (Lam.) Verdc.) Genotypes for Horse Gram Yellow Mosaic Virus Resistance.

Horse gram (Macrotyloma uniflorum (Lam.) Verdc.) is an under-utilized legume grown in India. It is a good source of protein, carbohydrates, dietary fiber, minerals, and vitamins. We screened 252 horse gram germplasm accessions for horse gram yellow mosaic virus resistance using the percent disease index and scaling techniques. The percentage values of highly resistant, moderately resistant, moderately susceptible, susceptible, and highly susceptible were 0.34, 13.89, 38.89, 46.43, and 0.34, respectively. Repetitive trials confirmed the host-plant resistance levels, and yield loss was assessed. The present disease index ranged from 1.2 to 72.0 and 1.2 to 73.0 during the kharif and rabi seasons of 2018, respectively. The maximum percent yield loss was noticed in the HS (75.0 -89.4), while HR possessed the minimum (1.2-2.0). The methanolic leaf extracts of highly resistant and highly susceptible genotypes with essential controls were subjected to gas chromatography-mass spectrometry analysis. Differential accumulation of metabolites was noticed, and a total of 81 metabolites representing 26 functional groups were identified. Both highly resistant and susceptible genotypes harbored eight unique classes, while ten biomolecules were common. The hierarchical cluster analysis indicated a distinct metabolite profile. Fold change in the common metabolites revealed an enhanced accumulation of sugars, alkanes, and carboxylic acids in the highly resistant genotype. The principal component analysis plots explained 93.7% of the variation. The metabolite profile showed a significant accumulation of three anti-viral (octadecanoic acid, diphenyl sulfone, and 2-Aminooxazole), one insecticidal (9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol), one antifeedant (cucurbitacin B), and six metabolites with unknown biological function in the highly resistant genotype.

Phone Camera Nano-Biosensor Using Mighty Sensitive Transparent Reusable Upconversion Paper.

Lycopene, a natural colorant and antioxidant with a huge growing market, is highly susceptible to photo/thermal degradation, which demands real-time sensors. Hence, here a transparent upconversion nanoparticles (UCNPs) strip having 30 mol % Yb, 0.1 mol % Tm, and ß-NaYF4 UCNPs, which shows an intense emission at 475 nm, has been developed. This strip has been found to be sensitive to lycopene with a detection limit as low as 10 nM using a smartphone camera, which is due to static quenching that is confirmed by the lifetime study. In comparison to previous paper strips, here the transparent strip has minimal scattering with maximum sensitivity in spite of not using any metal quenchers. An increase in strip hydrophobicity during the fabrication process complements the strip to selectively permeate and present an extraction-free substitute analysis for chromatography. Hydrophobicity endows the strip with the capability to reuse the strip with ∼100% luminescence recovery.

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.).

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3-4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l-1) and 2,4-D (0.5 mg l-1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l-1 TDZ in combination with auxin 0.5 mg l-1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers.

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.

Expression, Purification and Initial Characterization of Functional α1-Microglobulin (A1M) in Nicotiana benthamiana.

α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.

A non-classical route of efficient plant uptake verified with fluorescent nanoparticles and root adhesion forces investigated using AFM.

Classical plant uptake is limited to hydrophilic or water-dispersible material. Therefore, in order to test the uptake behaviour of hydrophobic particles, here, we tested the fate of hydrophobic particles (oleylamine coated Cu2-xSe NPs (CS@OA)) in comparison to hydrophilic particles (chitosan-coated Cu2-xSe NPs (CS@CH)) by treatment on the plant roots. Surprisingly, hydrophobic CS@OA NPs have been found to be ~ 1.3 times more efficient than hydrophilic CS@CH NPs in tomato plant root penetration. An atomic force microscopy (AFM) adhesion force experiment confirms that hydrophobic NPs experience non-spontaneous yet energetically favorable root trapping and penetration. Further, a relative difference in the hydrophobic vs. hydrophilic NPs movement from roots to shoots has been observed and found related to the change in protein corona as identified by two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) analysis. Finally, the toxicity assays at the give concentration showed that Cu2-xSe NPs lead to non-significant toxicity as compared to control. This technology may find an advantage in fertilizer application.

Transient expression and purification of ß-caryophyllene synthase in Nicotiana benthamiana to produce ß-caryophyllene in vitro.

The sesquiterpene ß-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, ß-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 ß-caryophyllene. In this study, a full-length cDNA of a new duplicated ß-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce ß-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 ß-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 ß-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.

Plant based production of myoglobin - a novel source of the muscle heme-protein.

Myoglobin is a heme-protein in the muscle of vertebrates with important functions in the oxygenation of tissues and as a regulator in nitric oxide signaling. Myoglobin from many species is also an important nutritional source of bioavailable iron. In this study, we have successfully produced human myoglobin in the leaves of Nicotiana benthamiana by transient expression using a viral vector delivered by Agrobacterium tumefaciens. Analyses confirmed that heme was incorporated and the protein was functional, with observed properties consistent with those of native myoglobins. A relatively high degree of purity could be achieved with low cost methods. The results show the high potential of plants as a production platform for heme proteins, a group of proteins of interest for iron nutrition applications and possible future pharmaceutical development.

Difference in the action spectra for UVR8 monomerisation and HY5 transcript accumulation in Arabidopsis.

The photoreceptor UV RESISTANCE LOCUS 8 (UVR8) activates photomorphogenic responses when plants are exposed to ultraviolet-B (UV-B) light. However, whereas the absorption spectrum of UVR8 peaks at 280 nm, action spectra for several photomorphogenic UV-B responses show maximal photon effectiveness at 290-300 nm. To investigate this apparent discrepancy we measured the effectiveness of UV wavelengths in initiating two responses in Arabidopsis: photoconversion of homodimeric UVR8 into the monomeric form, which is active in signaling, and accumulation of transcripts of the ELONGATED HYPOCOTYL 5 (HY5) transcription factor, which has a key role in UVR8-mediated responses. When purified UVR8 or Arabidopsis leaf extracts were exposed to UV light monomerisation was maximal at approximately 280 nm, which correlates with the UVR8 absorption spectrum. When intact plants were exposed to UV, monomerisation was most strongly initiated at approximately 290 nm, and this shift in maximal effectiveness could be explained by strong absorption or reflectance at 280 nm by leaf tissue. Notably, the action spectrum for accumulation of HY5 transcripts in the same leaf tissue samples used to assay UVR8 dimer/monomer status peaked at approximately 300 nm. Possible reasons for the difference in maximal photon effectiveness of UVR8 monomerisation and HY5 transcript accumulation in leaf tissue are discussed.

Effects of Overexpression of WRI1 and Hemoglobin Genes on the Seed Oil Content of Lepidium campestre.

The wild species field cress (Lepidium campestre), belonging to the Brassicaceae family, has potential to be developed into a novel oilseed- and catch crop, however, the species needs to be further improved regarding some important agronomic traits. One of them is its low oil content which needs to be increased. As far as we know there is no study aiming at increasing the oil content that has been reported in this species. In order to investigate the possibility to increase the seed oil content in field cress, we have tried to introduce the Arabidopsis WRINKLED1 (AtWRI1) or hemoglobin (Hb) genes from either Arabidopsis thaliana (AtHb2) or Beta vulgaris (BvHb2) into field cress with the seed specific expression. The hypothesis was that the oil content would be increased by overexpressing these target genes. The results showed that the oil content was indeed increased by up to 29.9, 20.2, and 25.9% in the transgenic lines expressing AtWRI1, AtHb2, and BvHb2, respectively. The seed oil composition of the transgenic lines did not significantly deviate from the seed oil composition of the wild type plants. Our results indicate that genetic modification can be used in this wild species for its fast domestication into a future economically viable oilseed and catch crop.

Studies on the expression of linalool synthase using a promoter-ß-glucuronidase fusion in transgenic Artemisia annua.

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 ß-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 652bp 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 LIS expression will most likely have little or no effect on artemisinin production.