Melanopsin-mediated amplification of cone signals in the human visual cortex.

The ambient daylight variation is coded by melanopsin photoreceptors and their luxotonic activity increases towards midday when colour temperatures are cooler, and irradiances are higher. Although melanopsin and cone photoresponses can be mediated via separate pathways, the connectivity of melanopsin cells across all levels of the retina enables them to modify cone signals. The downstream effects of melanopsin-cone interactions on human vision are however, incompletely understood. Here, we determined how the change in daytime melanopsin activation affects the human cone pathway signals in the visual cortex. A 5-primary silent-substitution method was developed to evaluate the dependence of cone-mediated signals on melanopsin activation by spectrally tuning the lights and stabilizing the rhodopsin activation under a constant cone photometric luminance. The retinal (white noise electroretinogram) and cortical responses (visual evoked potential) were simultaneously recorded with the photoreceptor-directed lights in 10 observers. By increasing the melanopsin activation, a reverse response pattern was observed with cone signals being supressed in the retina by 27% (p = 0.03) and subsequently amplified by 16% (p = 0.01) as they reach the cortex. We infer that melanopsin activity can amplify cone signals at sites distal to retinal bipolar cells to cause a decrease in the psychophysical Weber fraction for cone vision.

The role of melanopsin photoreception on visual attention linked pupil responses.

A decision during a visual task is marked by a task-evoked pupil dilation (TEPD) that is linked to the global cortical arousal state. Melanopsin expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) form the afferent pathway for this pupil response. Melanopsin activation also influences mood and arousal and increases activity in decision-making brain areas that receive direct ipRGC projections. Here, an optical photostimulation method controlled the excitations of all five photoreceptor classes in the human eye to isolate melanopsin-mediated photoreception. We hypothesised that the TEPD can be driven by directing active visual covert attention through the ipRGC pathway. When observers are completely certain of the stimulus presence, melanopsin-directed stimulation produces a TEPD of similar amplitude to a cone-directed stimulation, with their combination producing larger amplitudes. This dilation is satisfactorily modelled by linear addition with a higher melanopsin weighting in ipRGCs. Visual reaction times were longest in response to melanopsin-directed lights. Next, we asked whether the afferent photoreceptor input and decision certainty, controlled by priming the observer's a priori expectation, interact to drive the TEPD. Signal detection analysis showed that by fixing the predecision certainty (bias), the phasic arousal and TEPD amplitude vary with observer criterion (c') and sensitivity (d') but not with preferential activation of melanopsin. The signature feature of the melanopsin response during attention was a biphasic TEPD. We conclude that active covert attention can be modulated by visual information mediated via ipRGCs, but that phasic arousal responses marked using the TEPD are not increased by higher levels of melanopsin activation.

Multi-component redox system for selective and potent antineoplastic activity towards ovarian cancer cells.

Ovarian cancer is the deadliest gynecological cancer which rarely causes symptoms, and goes undetected until reaching the advanced stage of drug-resistant metastases. The cationic porphyrin meso-tetra(4-N-methylpyridyl)porphine (TMPyP) is a well-known photosensitizer (PS) used in photodyamic therapy (PDT) for curing cancer due to its strong affinity for DNA and high yield of reactive oxygen species (ROS) upon light activation. The practicality to irradiate tumor cells alone in the physiological system being slim (due to the close proximity of healthy cells and tumors), we looked for a variation in the PDT using a mixture of TMPyP with 1,5-dihydroxynapthalene (DHN) and Fe(III) ions at a mole ratio of 1:20:17 (drug combo) respectively in aqueous solution. The drug combo needs no photoactivation in H2O2 rich environment (mimicking the microenvironment of cancer/tumor), where it generates È®H and juglone, the latter being a known potent anticancer agent. In vitro studies of the drug combo in drug resistant and sensitive ovarian cancer cell lines showed drastic growth inhibition and cell death compared to normal epithelial cells. The drug combo provides an effective and non-invasive alternative to conventional PDT, exploiting the cytosolic carcinogenic H2O2 to produce an efficient anticancer treatment. The unique action of cancer-specific cytotoxicity arises from the redox chemistry involving activation of Fe(III) as the oxidizing agent to generate juglone, which utilizes the cytosolic ROS in cancer cells against itself.

Design and validation of a chart-based measure of the limits of spatial contrast sensitivity.

PURPOSE: Current chart-based tests of spatial contrast sensitivity (SCS) with fixed or narrow frequency ranges (≤18 cycles/°) cannot characterise the limits of spatial contrast vision. Here we present the design and validation of a chart-based measure of the spatial contrast envelope. METHODS: Following the principles of the standard visual acuity (Bailey-Lovie) and contrast sensitivity (Pelli-Robson) charts, a combined spatial-contrast and visual acuity chart was designed using a language-independent triangular symbol for a four-alternative forced-choice procedure plus chart rotation. Symbol frequencies ranged between 0.38 and 60 cycles/° spaced along 10 radial axes (0.55%-100% contrast). The chart was validated with reference to the Bailey-Lovie and Pelli-Robson charts; its reliability and sensitivity to changes in illumination, simulated cataract and blur was evaluated in healthy adults. RESULTS: The photopic SCS function could be measured in 5.5 ± 0.5 min; thresholding around the spatial contrast resolution limit reduced completion times to ~2 min. There was good agreement with high-contrast visual acuity (difference = 0.08 ± 0.02 logMAR) and contrast-sensitivity at 1.5 cycles/° (0.13 ± 0.06 logCS). Test-retest reliability was excellent at all spatial frequencies (ICC = 0.99). Mesopic illumination or simulated cataract caused a generalised SCS loss; myopic blur reduced high-frequency sensitivity. Spatial contrast sensitivity was independent of radial axis orientation (cardinal or oblique). CONCLUSIONS: The chart provides a time-efficient, reliable and inexpensive measure of SCS with applications in research and clinic for detecting subtle deficits in early stages of ocular and neurological conditions that often manifest at higher frequencies. It is sensitive to vision changes occurring in dim lighting and with simulated cataract and blur. The chart is available open-access for self-printing; contrast variation in print can be controlled through user calibration and/or establishing normative SCS functions using the theoretical values.

TEM study of chronic alcoholism effects on early carcinogenesis by probing the nanoscale structural alterations of cell nuclei.

Nanoscale structural alteration in the nuclei of cells with the progression of carcinogenesis is due to the rearrangements of the basic building blocks in the cell such as DNA, RNA, lipids, etc. Although epigenetic modifications underlie the development of cancer, exposure to carcinogenic chemicals such as alcohol also enhances the development of cancer. We report the effects of chronic alcoholism on early-carcinogenesis based on changes in the degree of nanoscale structural alterations (L d) in nuclei. For this, transmission electron microscopy (TEM) imaging of the nuclei of colonic cells is performed for the following four mouse models: control mice; chronic alcoholic mice treated with ethanol (i.e., EtOH mice); mice treated with colonic carcinogen azoxymethane (AOM) and dextran sulfate sodium (DSS) that induced colitis (i.e., AOM + DSS mice); and chronic alcoholic or EtOH treated mice, together with AOM and DSS treatment (i.e., AOM + DSS + EtOH mice). The disordered optical lattices are constructed from their respective TEM images of thin colonic cell nuclei and the L d values are calculated using the inverse participation ratio (IPR) technique from the spatially localized eigenfunctions of these lattices. Results show no significant difference in the average L d value of the colon cell nuclei of alcohol treated mice relative to its control [i.e., L d(C) ∼ L d(EtOH)]; however, an increase in the L d value of alcohol treated precancerous cells [i.e., L d(AOM + DSS + EtOH) > L d(AOM + DSS)], indicating that alcohol accelerates the early carcinogenic process.

Melanopsin hypersensitivity dominates interictal photophobia in migraine.

PURPOSE: To define the melanopsin and cone luminance retinogeniculate pathway contributions to photophobia in healthy controls and migraineurs. METHODS: Healthy controls and migraineurs were categorized according to the International Classification of Headache Disorders criteria. Photophobia was measured under full-field illumination using electromyography in response to narrowband lights spanning the melanopsin and cone luminance action spectra. Migraineurs were tested during their interictal headache-free period. Melanopsin-mediated post-illumination pupil responses quantified intrinsically photosensitive Retinal Ganglion Cell (ipRGC) function. RESULTS: A model combining the melanopsin and cone luminance action spectra best described photophobia thresholds in controls and migraineurs; melanopsin contributions were ∼1.5× greater than cone luminance. In the illumination range causing photophobia, migraineurs had lower photophobia thresholds (∼0.55 log units; p < 0.001) and higher post-illumination pupil response amplitudes (p = 0.03) than controls. CONCLUSION: Photophobia is driven by melanopsin and cone luminance inputs to the cortex via the retino-thalamocortical pathway. In migraineurs, lower photophobia thresholds reflect hypersensitivity of ipRGC and cone luminance pathways, with the larger and prolonged post-illumination pupil response amplitude indicative of a supranormal melanopsin response. Our findings inform artificial lighting strategies incorporating luminaires with low melanopsin excitation and photopic luminance to limit the lighting conditions leading to photophobia.

Optimizing methods to isolate melanopsin-directed responses.

The intrinsic melanopsin photoresponse may initiate visual signals that differ in spatiotemporal characteristics from the cone-opsin- and rhodopsin-mediated signals. Applying the CIE standard observer functions in silent-substitution methods can require individual differences in photoreceptor spectral sensitivities and pre-receptoral filtering to be corrected; failure to do so can lead to the intrusion of more sensitive cone processes with putative melanopsin-directed stimuli. Here we evaluate heterochromatic flicker photometry (HFP) and photoreceptor-directed temporal white noise as techniques to limit the effect of these individual differences. Individualized luminous efficiency functions (V(λ)) were compared to the CIE standard observer functions. We show that adapting chromaticities used in silent-substitution methods can deviate by up to 54% in luminance when estimated with the individual and standard observer functions. These deviations lead to inadvertent cone intrusions in the visual functions measured with melanopsin-directed stimuli. To eliminate the intrusions, individual HFP corrections are sufficient at low frequencies (∼1Hz) but temporal white noise is also required at higher frequencies to desensitize penumbral cones. We therefore recommend the selective application of individualized observer calibration and/or temporal white noise in silent-substitution paradigms when studying melanopsin-directed photoresponses.

Fertilization in flowering plants: an odyssey of sperm cell delivery.

KEY MESSAGE: In light of the available discoveries in the field, this review manuscript discusses on plant reproduction mechanism and molecular players involved in the process. Sperm cells in angiosperms are immotile and are physically distant to the female gametophytes (FG). To secure the production of the next generation, plants have devised a clever approach by which the two sperm cells in each pollen are safely delivered to the female gametophyte where two fertilization events occur (by each sperm cell fertilizing an egg cell and central cell) to give rise to embryo and endosperm. Each of the successfully fertilized ovules later develops into a seed. Sets of macromolecules play roles in pollen tube (PT) guidance, from the stigma, through the transmitting tract and funiculus to the micropylar end of the ovule. Other sets of genetic players are involved in PT reception and in its rupture after it enters the ovule, and yet other sets of genes function in gametic fusion. Angiosperms have come long way from primitive reproductive structure development to today's sophisticated, diverse, and in most cases flamboyant organ. In this review, we will be discussing on the intricate yet complex molecular mechanism of double fertilization and how it might have been shaped by the evolutionary forces focusing particularly on the model plant Arabidopsis.

Establishment of a novel method for the identification of fertilization defective mutants in Arabidopsis thaliana.

Plant reproduction is an extremely important phenomenon, as it is strongly associated with plant genetics and early development. Additionally, foundations of the reproductive system have direct implications on plant breeding and agriculture. Investigation of the functions of male and female gametophytes is critical since their fusion is required for seed formation. Although a large number of mutants have been generated to understand the functions of male and female gametophytes, only a small number of genes required for plant fertilization have been identified to date. This is because the screening method used previously required the dissection of siliques, and fertilization-specific mutants exhibiting semi-fertility (or ∼50% fertility) were difficult to identify. Here, we report a new efficient screening method for the identification of fertilization defective mutants in Arabidopsis thaliana using vanillin staining. This method is based on the pollen tube-dependent ovule enlargement morphology (POEM) phenomenon, which generates a partial seed coat within the ovule without fertilization. Using this method, we successfully identified 23 putative fertilization defective mutants in Arabidopsis.

Enhanced monoterpene emission in transgenic orange mint (Mentha × piperita f. citrata) overexpressing a tobacco lipid transfer protein (NtLTP1).

MAIN CONCLUSION: Overexpression of the tobacco lipid transfer protein (NtLTP1) gene in transgenic orange mint resulted in enhanced accumulation of monoterpenes in the cavity of head cells of glandular trichomes, which resulted in enhanced emission of monoterpenes from transgenic orange mints. Plants in the genus Mentha (Lamiaceae) produce volatile oils that accumulate in peltate glandular trichomes in the aerial parts of plants. A lipid transfer protein (NtLTP1) in tobacco showed glandular trichome-specific expression and supported the secretion of diterpenoid lipids from head cells of glandular trichomes (Choi et al., Plant J 70:480-491,2012). Here, we constructed transgenic orange mint (Mentha × piperita f. citrata) overexpressing the tobacco NtLTP1 gene via Agrobacterium-mediated transformation. Transgenic lines of orange mint overexpressing NtLTP1 were confirmed by genomic PCR and RT-PCR. Immunoblotting analysis using an NtLTP1 polyclonal antibody showed clear dark spots at the position of the lipid exudates from tobacco glandular trichomes and the squeezed out lipids from the glandular trichomes of transgenic orange mint. Heads of glandular trichomes in transgenic plants overexpressing the NtLTP1 gene showed a larger diameter than those of the wild-type control. The enhanced size of trichome heads in transgenic orange mint was confirmed by scanning electron microscopy. Volatile components were extracted from wild-type and transgenic orange mint by solid-phase microextraction (SPME) and analyzed by headspace-gas chromatography-mass spectrometry (HS/GC/MS). Linalyl acetate was the most abundant component among the eleven identified monoterpenes in the volatile compounds extracted from both the wild-type and transgenic lines of orange mint. Overexpression of NtLTP1 in transgenic orange mint plants resulted in enhanced emission of volatile monoterpenoids compared with that of volatile monoterpenoids in the wild-type control plants.

Studying nanoscale structural alterations in cancer cells to evaluate ovarian cancer drug treatment, using transmission electron microscopy imaging.

Understanding nanoscale structural changes can provide information about the physical state of cells/tissues. It has now been shown that increases in nanoscale structural alterations are associated with the progress of carcinogenesis in most cancer cases, including early carcinogenesis. Anti-cancerous therapies are designed to inhibit the growth of cancer cells; however, it is challenging to detect the efficacy of such drugs in the early stages of treatment. A unique method of assessing the impact of anti-cancerous drugs on cancerous cells/tissues is to probe the nanoscale structural alterations. In this paper, we study the effect of different anti-cancerous drugs on ovarian tumorigenic cells, using their nanoscale structural alterations as a biomarker. Transmission electron microscopy (TEM) imaging on thin cell sections is performed to obtain their nanoscale structures. The degree of nanoscale structural alterations of tumorigenic cells and anti-cancerous drug treated tumorigenic cells are quantified by using the recently developed inverse participation ratio (IPR) technique. Results show an increase in the degree of nanoscale fluctuations in tumorigenic cells relative to non-tumorigenic cells; then a near-reversal of the degree of fluctuation in tumorigenic cells to that in non-tumorigenic cells, following anti-cancerous drug treatment. These results support that the effect of anti-cancerous drugs in cancer treatment can be quantified by using the degree of nanoscale fluctuations in the cells via TEM imaging. Potential applications of the technique for cancer treatment are also discussed.

The accuracy of artificial and natural light measurements by actigraphs.

Actigraphs are the reference standard for measuring light exposure in human non-laboratory experiments due to their portability and long battery lives. However, actigraphs typically have a limited illuminance operating range not representative of real-world conditions, and for many actigraphs, the accuracy of their light measurement has not been verified independently. We assessed the illuminances recorded by Activinsights GENEActiv Original and Philips Actiwatch 2 actigraphs in comparison to a calibrated, laboratory-standard photometer, under both artificial light-emitting diode (LED) and natural sunlight illuminations that might be encountered by a person under real-world conditions. We show that in response to ~20,000 lux white LED light, the GENEActiv and Actiwatch 2 underestimate illuminance by recording 50% and 25% of the true value, respectively. Under ~30,000 lux sunlight, the GENEActiv readily saturates whereas the Actiwatch 2 reports ~46% of the true illuminance. These underestimations are highly linear and we provide correction factors to estimate the illuminance levels of the ambient environment measured by the actigraphs. We also evaluate the application of neutral density filters for extending the operating range of both devices in natural sunlight illuminations (as high as 30,000 lux during our measurements) and demonstrate that this may be a viable approach for increasing the operating range of the Actiwatch 2 but not the GENEActiv. We conclude that both actigraphs provide good performance in monitoring the temporal patterning of light, whereas the absolute illuminance values require correction to accurately evaluate the effects of light intensity on human health and behaviours.

Rhodopsin and melanopsin contributions to human brightness estimation.

We examined the contributions of rhodopsin and melanopsin to human brightness estimation under dim lighting. Absolute brightness magnitudes were estimated for full-field, rhodopsin-, or melanopsin-equated narrowband lights (${\lambda _{\rm max}}:\;{462}$λmax:462, 499, 525 nm). Our data show that in scotopic illumination ($ - {5.1}$-5.1 to $ - {3.9}\;{\log}\;\unicode{x00B5} {\rm Watts}\cdot{\rm cm}^{ - 2}$-3.9logµWatts⋅cm-2), the perceptual brightness estimates of rhodopic irradiance-equated conditions are independent of their corresponding melanopic irradiance, whereas brightness estimates with melanopic irradiance-equated conditions increase with increasing rhodopic irradiance. In mesopic illumination ($ - {3.4}$-3.4 to $ - {1.9}\;{\log}\;\unicode{x00B5} {\rm Watts}\cdot{\rm cm}^{ - 2}$-1.9logµWatts⋅cm-2), the brightness estimates with both lighting conditions increase with increasing rhodopic or melanopic irradiances. Rhodopsin activation therefore entirely signals scotopic brightness perception and plateaus in mesopic illumination where intrinsic melanopsin contributions become first evident. We infer that all photoreceptor signals are transmitted to higher visual centers for representing scene brightness in scotopic and mesopic illumination through both conventional and melanopsin ganglion cell pathways.

Spectroscopic Study on Pseudomonas Aeruginosa Biofilm in the Presence of the Aptamer-DNA Scaffolded Silver Nanoclusters.

We report the effectiveness of silver nanocluster (Ag-NC) against the biofilm of Pseudomonas aeruginosa (PA). Two DNA aptamers specific for PA and part of their sequences were chosen as templates for growing the Ag-NC. While circular dichroism (CD) studies determined the presence of secondary structures, UV/Vis absorption, and fluorescence spectroscopic studies confirmed the formation of the fluorescent Ag-NC on the DNA templates. Furthermore, mesoscopic physics-based partial wave spectroscopy (PWS) was used to analyze the backscattered light signal that can detect the degree of nanoscale mass density/refractive index fluctuations to identify the biofilm formation, comparatively among the different aptamers with respect to the control sample. The importance of the secondary structure of the aptamer DNA in targeting, successfully binding with the cells and delivering the Ag-NC, is evidenced by the decrease in disorder strength (Ld) of the Ag-NC treated samples compared to the untreated PA cells, which showed the abundance of higher Ld in the PWS studies. The higher Ld value attributed to the higher mass density fluctuations and the formation of biofilm. We envision this study to open a new avenue in using a powerful optical microscopic technique like PWS in detection, and DNA aptamer enclosed silver nanoclusters to prevent biofilms for opportunist pathogens like Pseudomonas aeruginosa.

Lipid Transfer Proteins (AaLTP3 and AaLTP4) Are Involved in Sesquiterpene Lactone Secretion from Glandular Trichomes in Artemisia annua.

In Artemisia annua plants, glandular trichomes (GTs) are responsible for the biosynthesis and secretion of sesquiterpene lactones including artemisinin/arteannuin B. Nonspecific lipid transfer proteins (LTPs) in plants bind and carry lipid molecules across the cell membrane and are also known as secretary proteins. Interestingly, the transcripts of LTP genes are exceptionally abundant in the GTs of A. annua. In the present study, we isolated two trichome-specific LTP genes (AaLTP3 and AaLTP4) from a Korean ecotype of A. annua. AaLTP3 was expressed abundantly in shoots, whereas AaLTP4 was expressed in flowers. The GUS signal driven by the AaLTP3 or AaLTP4 promoter in transgenic A. annua plants revealed that the AaLTP3 promoter was active on hair-like non-GTs and that the AaLTP4 promoter was active on GTs. Analysis of enhanced cyan fluorescent protein (ECFP) fluorescence fused with the AaLTP3 or AaLTP4 protein in transgenic tobacco revealed that ECFP florescence was very bright on secreted lipids of long GTs. Moreover, the florescence was also bright on the head cells of short trichomes and their secreted granules. Immunoblotting analysis of GT exudates in petioles of A. annua revealed a strong positive signal against the AaLTP4 antibody. Overexpression of AaLTP3 or AaLTP4 in transgenic A. annua plants resulted in enhanced production of sesquiterpene lactones (arteannuin B, artemisinin, dihydroartemisinic acid and artemisinic acid) compared with those of wild type. The present study shows that LTP genes (AaLTP3 or AaLTP4) play important roles in the sequestration and secretion of lipids in GTs of A. annua, which is useful for the enhanced production of sesquiterpene lactones by genetic engineering.

Functional characterization of an oxidosqualene cyclase (PdFRS) encoding a monofunctional friedelin synthase in Populus davidiana.

MAIN CONCLUSION: An oxidosqualene cyclase (PdFRS) from Populus davidiana was characterized as a monofunctional friedelin synthase by its heterologous expression in yeast and overexpression in plants. Triterpenes are one of the largest classes of plant chemical compounds composed of three terpene units, which form the basic skeleton of all sterols and saponins. Friedelin (friedelan-3-one), a pentacyclic triterpene, occurs in many plant families and is particularly present in rich amounts in cork tissues from trees. The biosynthesis of friedelin occurs through the oxidosqualene cyclase (OSC) enzyme that generates friedelin from 2,3-oxidosqualene after the maximum rearrangement of a triterpene skeleton. Populus davidiana is called Korean aspen and grows in northern East Asia. From 57,322 unique sequences generated from the P. davidiana transcriptome database, one complete coding sequence (PdFRS) was obtained from a contig, which showed 74% identity to Betula platyphylla ß-amyrin synthase and 73% identity with friedelin synthase from Maytenus ilicifolia. The open reading frame (ORF) region of the PdFRS sequence was 2280 bp long and composed a 759 amino acid protein with a predicted molecular mass of 87.81 kDa. qPCR analysis revealed that methyl jasmonate treatments strongly upregulated PdFRS gene expression and resulted in enhanced friedelin accumulation in leaves. Heterologous expression of the PdFRS gene in yeast resulted in the production of friedelin triterpene as a single product, which was confirmed by comparison with the mass fragmentation pattern from an authentic friedelin standard by GC/MS analysis. Transgenic P. davidiana overexpressing the PdFRS gene was constructed via Agrobacterium-mediated transformation. Overexpression of PdFRS in transgenic P. davidiana lines resulted in enhanced friedelin production.

Cone and melanopsin contributions to human brightness estimation: reply.

Our analytical description of full-field brightness perception data [J. Opt. Soc. Am. A35, B19 (2018)JOAOD60740-323210.1364/JOSAA.35.000B19] with contributions from cone luminance and melanopsin expressing intrinsically photosensitive retinal ganglion cells has been extended [J. Opt. Soc. Am. A35, 1780 (2018)JOAOD60740-323210.1364/JOSAA.35.001780] to include S-cones through a blue-yellow opponent channel. We welcome this reanalysis and provide a few remarks on the approach.

Cone and melanopsin contributions to human brightness estimation.

We determined the contributions of cone and melanopsin luminance signaling to human brightness perception. The absolute brightness of four narrowband primary lights presented in a full-field Ganzfeld was estimated in two conditions, either cone luminance-equated (186.7-1,867.0 cd·m-2) or melanopsin luminance-equated (31.6-316.3 melanopsin cd·m-2). We show that brightness estimations for each primary light follow an approximately linear increase with increasing cone or melanopsin luminance (in log units), but are not equivalent for primary lights equated with either cone or melanopsin luminance. Instead, brightness estimations result from a combined interaction between cone and melanopsin signaling. Analytical modeling with wavelength-dependent coefficients signifies that melanopsin luminance positively correlates with brightness magnitudes, and the cone luminance has two contribution components, one that is additive to melanopsin luminance and a second that is negative, implying an adaptation process. These results provide a new framework for evaluating the physiological basis of brightness perception and have direct practical applications for the development of energy-efficient light sources.

Heterologous production of a ginsenoside saponin (compound K) and its precursors in transgenic tobacco impairs the vegetative and reproductive growth.

MAIN CONCLUSION: Production of compound K (a ginsenoside saponin) and its precursors in transgenic tobacco resulted in stunted growth and seed set failure, which may be caused by strong autotoxicity of heterologously produced phytochemicals against the tobacco itself. Panax ginseng roots contain various saponins (ginsenosides), which are major bioactive compounds. A monoglucosylated saponin, compound K (20-O-(ß-D-glucopyranosyl)-20(S)-protopanaxadiol), has high medicinal and cosmetic values but is present in undetectable amounts in naturally grown ginseng roots. The production of compound K (CK) requires complicated deglycosylation of ginsenosides using physicochemical and/or enzymatic degradation. In this work, we report the production of CK in transgenic tobacco by co-overexpressing three genes (PgDDS, CYP716A47 and UGT71A28) isolated from P. ginseng. Introduction and expression of the transgenes in tobacco lines were confirmed by genomic PCR and RT-PCR. All the lines of transgenic tobacco produced CK including its precursors, protopanaxadiol and dammarenediol-II (DD). The concentrations of CK in the leaves ranged from 1.55 to 2.64 µg/g dry weight, depending on the transgenic line. Interestingly, production of CK in tobacco brought stunted plant growth and gave rise to seed set failure. This seed set failure was caused by both long-styled flowers and abnormal pollen development in transgenic tobacco. Both CK and DD treatments highly suppressed in vitro germination and tube growth in wild-type pollens. Based on these results, metabolic engineering for CK production in transgenic tobacco was successfully achieved, but the production of CK and its precursors in tobacco severely affects vegetative and reproductive growth due to the cytotoxicity of phytochemicals that are heterologously produced in transgenic tobacco.