Inhibitor Of Carotenoid Biosynthesis Research Articles

Uncovering the Hidden Potential of Phytoene Production by the Fungus Blakeslea trispora.

Phytoene is an uncommon linear carotene within the carotenoid group as it is colorless due to its short chromophore. Recent research constitutes a relatively new area which has emerged from phytoene's importance as a major dietary carotenoid promoting health and appearance. Its resources point to the potential of biotechnological production systems. Our work has been designed to study the efficacy of two colored carotenoid biosynthesis inhibitors, diphenylamine and 2-methyl imidazole, and one sterol biosynthesis inhibitor, terbinafine, to modify the metabolic flux in mated cultures of Blakeslea trispora to achieve maximum phytoene production. Bioprocess kinetics optimized by response surface methodology and monitored by high-performance liquid chromatography revealed maximum phytoene content (5.02 mg/g dry biomass) and yield (203.91 mg/L culture medium) comparable or even higher than those reported for other potent phytoene microbial producers. The in vivo antioxidant activity of phytoene-rich carotenoid extract from fungal cells was also considered and discussed.

Carotenoid biosynthesis is associated with low-temperature adaptation in Rhodosporidium kratochvilovae

BackgroundLow temperatures greatly limit the growth of microorganisms. Low-temperature adaptation in microorganisms involves multiple mechanisms. Carotenoids are naturally occurring lipid-soluble pigments that act as antioxidants and protect cells and tissues from the harmful effects of free radicals and singlet oxygen. However, studies on the regulation of carotenoid biosynthesis at low temperatures in microorganisms are limited. In this study, we investigated the correlation between carotenoids and low-temperature adaptation in the cold-adapted strain of Rhodosporidium kratochvilovae YM25235.ResultsCarotenoid biosynthesis in YM25235 was inhibited by knocking out the bifunctional lycopene cyclase/phytoene synthase gene (RKCrtYB) using the established CRISPR/Cas9 gene-editing system based on endogenous U6 promoters. The carotenoids were extracted with acetone, and the content and composition of the carotenoids were analyzed by spectrophotometry and HPLC. Then, the levels of reactive oxygen species (ROS) and the growth rate in YM25235 were determined at a low temperature. The results indicated that the carotenoid biosynthesis and ROS levels were increased in the YM25235 strain at a low temperature and inhibition of carotenoid biosynthesis was associated with higher ROS levels and a significant decrease in the growth rate of YM25235 at a low temperature.ConclusionsThe regulation of carotenoid biosynthesis was associated with low-temperature adaptation in YM25235. Our findings provided a strong foundation for conducting further studies on the mechanism by which YM25235 can adapt to low-temperature stress.

Plastid methylerythritol phosphate pathway participates in the hypersensitive response-related cell death in Nicotiana benthamiana.

Programmed cell death (PCD), a characteristic feature of hypersensitive response (HR) in plants, is an important cellular process often associated with the defense response against pathogens. Here, the involvement of LytB, a gene encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase that participates in the final step of the plastid methylerythritol phosphate (MEP) pathway, in plant HR cell death was studied. In Nicotiana benthmiana plants, silencing of the NbLytB gene using virus-induced gene silencing (VIGS) caused plant growth retardation and albino leaves with severely malformed chloroplasts. In NbLytB-silenced plants, HR-related cell death mediated by the expression of either the human proapoptotic protein gene Bax or an R gene with its cognate Avr effector gene was inhibited, whereas that induced by the nonhost pathogen Pseudomonas syringae pv. syringae 61 was enhanced. To dissect the isoprenoid pathway and avoid the pleiotropic effects of VIGS, chemical inhibitors that specifically inhibit isoprenoid biosynthesis in plants were employed. Treatment of N. benthamiana plants with fosmidomycin, a specific inhibitor of the plastid MEP pathway, effectively inhibited HR-related PCD, whereas treatment with mevinolin (a cytoplasmic mevalonate pathway inhibitor) and fluridone (a carotenoid biosynthesis inhibitor) did not. Together, these results suggest that the MEP pathway as well as reactive oxygen species (ROS) generation in the chloroplast play an important role in HR-related PCD, which is not displaced by the cytosolic isoprenoid biosynthesis pathway.

Mixture Toxicity of Herbicides with Dissimilar Modes of Action to Myriophyllum spicatum.

Considering the vital role of rooted macrophytes in the aquatic ecosystem, validating assumptions on the interactive effects of herbicides with different modes of action at an environmentally relevant mixture ratio is necessary. We investigated the effects of diflufenican (a carotenoid biosynthesis inhibitor) and iodosulfuron-methyl-sodium (IMS; an acetolactate synthase inhibitor) in a 14-day growth inhibition experiment with Myriophyllum spicatum, wherein single compounds and their combination were tested in parallel (n = 84). The assessment was done using three different methods: significance testing, model deviation ratio (MDR), and mixture interaction factor (MIF). Interactions relative to both concentration addition and independent action were assessed via significance testing. This revealed that diflufenican and IMS acted antagonistically relative to both models for fresh weight and total shoot length (p < 0.05) and that there was slight synergism for the number of side shoots (p < 0.001) relative to concentration addition. The MDR and MIF can only assess interactions relative to the concentration addition model. According to MDR, the mixture appeared to show no interaction (neither antagonistic nor synergistic), whereas the MIF method revealed that the compounds acted antagonistically for fresh weight and that there was a slight synergism for total shoot length and number of side shoots. We conclude that inferences about mixture toxicity interactions are method- and endpoint-dependent, which can have implications for regulatory mixtures assessment. Environ Toxicol Chem 2022;41:2209-2220. © 2022 SETAC.

Tetrapyrrole biosynthesis pathway regulates plastid-to-nucleus signaling by controlling plastid gene expression in plants

Plastid-to-nucleus retrograde signaling coordinates nuclear gene expression with chloroplast developmental status and is essential for the photoautotrophic lifestyle of plants. Previous studies have established that tetrapyrrole biosynthesis (TPB) and plastid gene expression (PGE) play essential roles in plastid retrograde signaling during early chloroplast biogenesis; however, their functional relationship remains unknown. In this study, we generated a series of rice TPB-related gun (genome uncoupled) mutants and systematically analyzed their effects on nuclear and plastid gene expression under normal conditions or when subjected to treatments with norflurazon (NF; a noncompetitive inhibitor of carotenoid biosynthesis) and/or lincomycin (Lin; a specific inhibitor of plastid translation). We show that under NF treatment, expression of plastid-encoded polymerase (PEP)-transcribed genes is significantly reduced in the wild type but is derepressed in the TPB-related gun mutants. We further demonstrate that the derepressed expression of PEP-transcribed genes may be caused by increased expression of the PEP core subunit and nuclear-encoded sigma factors and by elevated copy numbers of plastid genome per haploid genome. In addition, we show that expression of photosynthesis-associated nuclear genes (PhANGs) and PEP-transcribed genes is correlated in the rice TPB-related gun mutants, with or without NF or Lin treatment. A similar correlation between PhANGs and PGE is also observed in the Arabidopsis gun4 and gun5 mutants. Moreover, we show that increased expression of PEP-transcribed plastid genes is necessary for the gun phenotype in NF-treated TPB-related gun mutants. Further, we provide evidence that these TPB-related GUN genes act upstream of GUN1 in the regulation of retrograde signaling. Taken together, our results suggest that the TPB-related GUN genes control retrograde plastid signaling by regulating the PGE-dependent retrograde signaling pathway.

A foliar pigment-based bioassay for interrogating chloroplast signalling revealed that carotenoid isomerisation regulates chlorophyll abundance

BackgroundSome plastid-derived metabolites can control nuclear gene expression, chloroplast biogenesis, and chlorophyll biosynthesis. For example, norflurazon (NFZ) induced inhibition of carotenoid biosynthesis in leaves elicits a protoporphyrin IX (Mg-ProtoIX) retrograde signal that controls chlorophyll biosynthesis and chloroplast development. Carotenoid cleavage products, known as apocarotenoids, also regulate plastid development. The key steps in carotenoid biosynthesis or catabolism that can regulate chlorophyll biosynthesis in leaf tissues remain unclear. Here, we established a foliar pigment-based bioassay using Arabidopsis rosette leaves to investigate plastid signalling processes in young expanding leaves comprising rapidly dividing and expanding cells containing active chloroplast biogenesis.ResultsWe demonstrate that environmental treatments (extended darkness and cold exposure) as well as chemical (norflurazon; NFZ) inhibition of carotenoid biosynthesis, reduce chlorophyll levels in young, but not older leaves of Arabidopsis. Mutants with disrupted xanthophyll accumulation, apocarotenoid phytohormone biosynthesis (abscisic acid and strigolactone), or enzymatic carotenoid cleavage, did not alter chlorophyll levels in young or old leaves. However, perturbations in acyclic cis-carotene biosynthesis revealed that disruption of CAROTENOID ISOMERASE (CRTISO), but not ZETA-CAROTENE ISOMERASE (Z-ISO) activity, reduced chlorophyll levels in young leaves of Arabidopsis plants. NFZ-induced inhibition of PHYTOENE DESATURASE (PDS) activity caused higher phytoene accumulation in younger crtiso leaves compared to WT indicating a continued substrate supply from the methylerythritol 4-phosphate (MEP) pathway.ConclusionThe Arabidopsis foliar pigment-based bioassay can be used to differentiate signalling events elicited by environmental change, chemical treatment, and/or genetic perturbation, and determine how they control chloroplast biogenesis and chlorophyll biosynthesis. Genetic perturbations that impaired xanthophyll biosynthesis and/or carotenoid catabolism did not affect chlorophyll biosynthesis. The lack of CAROTENOID ISOMERISATION reduced chlorophyll accumulation, but not phytoene biosynthesis in young leaves of Arabidopsis plants growing under a long photoperiod. Findings generated using the newly customised foliar pigment-based bioassay implicate that carotenoid isomerase activity and NFZ-induced inhibition of PDS activity elicit different signalling pathways to control chlorophyll homeostasis in young leaves of Arabidopsis.

What makes a molecule a pre- or a post-herbicide - how valuable are physicochemical parameters for their design?

Pre‐emergence herbicides are taken up by seeds before germination and by roots, hypocotyls, cotyledons, coleoptiles or leaves before emergence, whereas post‐emergence herbicides are taken up primarily by foliage and stems. Most modern pre‐emergence herbicides are lipophilic, but post‐emergence herbicides may be lipophilic or hydrophilic. The metabolic conversion of herbicides to inactive or active metabolites after plant uptake is of major importance for some compound classes. Several herbicides are proherbicides as for example some acetyl‐coenzyme A carboxylase (ACCase)‐inhibitors. The physicochemical characteristics of proherbicides and herbicides are usually unrelated. A major role can be attributed to the site of action at a cellular level. A great number of herbicides such as photosystem II (PS II)‐inhibitors, protoporphyrinogen oxidase (PPO)‐inhibitors or carotenoid biosynthesis inhibitors require light for activity. Others, such as cellulose‐biosynthesis and mitotic inhibitors seem to be primarily active in belowground organs. Several lipophilic barriers against the uptake of xenobiotics exist in aboveground and belowground plant parts. The relevance of these barriers needs, however, further clarification. Uptake and translocation models are valuable tools for the explanation of the potential movement of compounds. Many factors other than uptake and translocation have, however, to be considered for the design of herbicides. For post‐emergence herbicides, ultraviolet (UV) light stability, stability in formulations, and mixability with other agrochemicals have to be kept in mind while, in addition to the aforementioned factors soil interaction plays a major role for pre‐emergence herbicides. In our opinion, general physicochemical characteristics of pre‐ or post‐emergence herbicides do, unfortunately not exist yet. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot.

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.

High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance.

Microalgae represent a carbon-neutral source of bulk biomass, for extraction of high-value compounds and production of renewable fuels. Due to their high metabolic activity and reproduction rates, species of the genus Chlorella are highly productive when cultivated in photobioreactors. However, wild-type strains show biological limitations making algal bioproducts expensive compared to those extracted from other feedstocks. Such constraints include inhomogeneous light distribution due to high optical density of the culture, and photoinhibition of the surface-exposed cells. Thus, the domestication of algal strains for industry makes it increasingly important to select traits aimed at enhancing light-use efficiency while withstanding excess light stress. Carotenoids have a crucial role in protecting against photooxidative damage and, thus, represent a promising target for algal domestication. We applied chemical mutagenesis to Chlorella vulgaris and selected for enhanced tolerance to the carotenoid biosynthesis inhibitor norflurazon. The NFR (norflurazon-resistant) strains showed an increased carotenoid pool size and enhanced tolerance towards photooxidative stress. Growth under excess light revealed an improved carbon assimilation rate of NFR strains with respect to WT. We conclude that domestication of Chlorella vulgaris, by optimizing both carotenoid/chlorophyll ratio and resistance to photooxidative stress, boosted light-to-biomass conversion efficiency under high light conditions typical of photobioreactors. Comparison with strains previously reported for enhanced tolerance to singlet oxygen, reveals that ROS resistance in Chlorella is promoted by at least two independent mechanisms, only one of which is carotenoid-dependent.

A herbicide resistance risk assessment for weeds in wheat and barley crops in New Zealand.

We estimated the risk of selecting for herbicide resistance in 101 weed species known to occur in wheat and barley crops on farms in New Zealand. A protocol was used that accounts for both the risk that different herbicides will select for resistance and each weed's propensity to develop herbicide resistance based on the number of cases worldwide. To provide context we documented current herbicide use patterns. Most weeds (55) were low-risk, 30 were medium-risk and 16 high-risk. The top ten scored weeds were Echinochloa crus-galli, Poa annua, Lolium multiflorum, Erigeron sumatrensis, Raphanus raphanistrum, Lolium perenne, Erigeron bonariensis, Avena fatua, Avena sterilis and Digitaria sanguinalis. Seven out of ten high-risk weeds were grasses. The most used herbicides were synthetic auxins, an enolpyruvylshikimate-phosphate synthase inhibitor, acetolactate synthase (ALS) inhibitors, carotenoid biosynthesis inhibitors, and long-chain fatty acid inhibitors. ALS-inhibitors were assessed as posing the greatest risk for more species than other modes-of-action. Despite pre-emergence herbicides being known to delay resistance, New Zealand farmers only applied flufenacet and terbuthlazine with high frequency. Based on our analysis, surveys for herbicide-resistant species should focus on the high-risk species we identified. Farmer extension efforts in New Zealand should address resistance evolution in cropping weeds.

Assembly of the LH2 Light-Harvesting Complexes of Thiorhodospira sibirica with Different Carotenoid Levels

Regardless of cultivation conditions, two types of LH2 complexes are always assembled in photosynthetic membranes of Thiorhodospira (T.) sibirica: short-wave B800−830 and long-wave B800−850. The present work studied the effect of qualitative and quantitative content of carotenoids on assembling of these complexes. Diphenylamine (DPA), an inhibitor of carotenoid biosynthesis, was used to decrease the content of carotenoids in photosynthetic membranes and the complexes. In the cells of T. sibirica strain A12T, inhibition of carotenoid biosynthesis ranged from 40 to 50%. Complete suppression of carotenoid biosynthesis was achieved only once, which resulted in absorption spectrum changes of the DPA-LH2 complexes and lower stability of their structure; they were often destroyed during the isolation. The use of DPA with the cells of T. sibirica strain Kir-3 was more efficient. From inhibited cells of this strain, B800−850 and B800−830 complexes were isolated, in which the carotenoid content decreased to 80, 50, and 10%. Their spectral characteristics in the near infrared region were independent of the carotenoid quantity in the complexes and were similar to those of native complexes of the wild-type. At higher DPA concentration in the cultivation medium, accompanied by a decrease of the overall carotenoid content, such carotenoids of the earlier stages of biosynthesis as neurosporene, ζ-carotene, and their hydroxy derivatives, were found in the complexes. The efficiency of energy transfer from carotenoids to bacteriochlorophyll in all LH2 complexes was not shown to depend on the composition and quantity of carotenoids in the samples and was ~40%. Since the near-infrared spectrum of the LH2-complex of the B800−850 type was similar to that of the B800−850 complexes from Rhodospirillum molishianum, with 8 pairs of α/β-heterodimers forming the basis of their structure, it is possible to assume that the structure of the studied B800−850 complex from T. sibirica also consists of 8 pairs of α/β-heterodimers. A single carotenoid molecule is known to occur per the α/β-heterodimer; therefore, on average less than one carotenoid molecule (8 × 0.1 = 0.8) occurs per an LH2 complex from T.sibirica with 10% carotenoid content. This implies that the population of LH2 complexes contains both carotenoidless LH2 complexes and the complexes containing one or more carotenoid molecules. Obviously, LH2 complexes of both types are assembled regardless of the quantity of carotenoids per a complex, and carotenoids are not the required component for their correct assembly in vivo.

Functional analysis of mTERF5 and mTERF9 contribution to salt tolerance, plastid gene expression and retrograde signalling in Arabidopsis thaliana.

We previously showed that Arabidopsis mda1 and mterf9 mutants, defective in the chloroplast-targeted mitochondrial transcription termination factors mTERF5 and mTERF9, respectively, display altered responses to abiotic stresses and abscisic acid (ABA), as well as perturbed development, likely through abnormal chloroplast biogenesis. To advance the functional analysis of mTERF5 and mTERF9, we obtained and characterized overexpression (OE) lines. Additionally, we studied genetic interactions between sca3-2, affected in the plastid-RNA polymerase RpoTp, and the mda1-1 and mterf9 mutations. We also investigated the role of mTERF5 and mTERF9 in plastid translation and plastid-to-nucleus signalling. We found that mTERF9 OE reduces salt and ABA tolerance, while mTERF5 or mTERF9 OE alter expression of nuclear and plastid genes. We determined that mda1-1 and mterf9 mutations genetically interact with sca3-2. Further, plastid 16S rRNA levels were reduced in mda1-1 and mterf9 mutants, and mterf9 was more sensitive to chemical inhibitors of chloroplast translation. Expression of the photosynthesis gene LHCB1, a retrograde signalling marker, was differentially affected in mda1-1 and/or mterf9 compared to wild-type Col-0, after treatments with inhibitors of carotenoid biosynthesis (norflurazon) or chloroplast translation (lincomycin). Moreover, mterf9, but not mda1-1, synergistically interacts with gun1-1, defective in GUN1, a central integrator of plastid retrograde signals. Our results show that mTERF9, and to a lesser extent mTERF5, are negative regulators of salt tolerance and that both genes are functionally related to RpoTp, and that mTERF9 is likely required for plastid ribosomal stability and/or assembly. Furthermore, our findings support a role for mTERF9 in retrograde signalling.

Genome uncoupled (gun) phenotype is associated with root growth repression in Arabidopsis seedlings grown on lincomycin

«Genome uncoupled» (gun) is a molecular phenotype manifesting in a high expression of a number of nuclear photosynthesis associated genes in plants with arrested chloroplast biogenesis. The chloroplast biogenesis arrest could be achieved by growing of plants in presence of lincomycin (plastid translation inhibitor) or norflurazon (carotenoid biosynthesis inhibitor). We have found that Arabidopsis thaliana gun1-1gun5-1 double mutant seedlings with gun phenotype associated with both lincomycin and norflurazon demonstrate a repressed root growth when germinated on lincomycin but not norflurazon-containing media. Statistical analysis has demonstrated a high negative correlation between root length and gun phenotype evaluated on both LHCB1.2 and HEMA1 genes expression in three gun mutants of Arabidopsis: gun1-1, cch1-1 and the double mutant gun1-1gun5-1. Our results demonstrate that regulatory signals of plastid origin might participate in root development.

The LH2 Complexes (B800-850 and B800-830) Are Assembled in the Cells of the Sulfur Bacterium Thiorhodospira sibirica Strain Kir-3 without Carotenoids.

The results of assembling the light-harvesting complexes in the cells of the purple sulfur bacterium Thiorhodospira (T.) sibirica strain Kir-3 while suppressing the biosynthesis of carotenoids with diphenylamine (DPA) were studied. LH2 complexes (B800-850 and B800-830) with different carotenoid composition were isolated from the cells obtained. Maximum inhibition of carotenoid biosynthesis (~90% of the control) was reached at an inhibitor concentration of 53.25 μM (9 mg/L). It was established that changes in the qualitative and quantitative composition of carotenoids do not affect the assembly of B800-830 and B800-850 complexes. It is assumed that, in the population of DPA-LH2 complexes from T. sibirica strain Kir-3, both the carotenoidless complexes and the complexes containing one or two carotenoid molecules can be assembled. These results support the hypothesis that carotenoids are not required for assembling B800-850 and B800-830 complexes.

TILLER ANGLE CONTROL 1 modulates plant architecture in response to photosynthetic signals.

Light serves as an important environmental cue in regulating plant architecture. Previous work had demonstrated that both photoreceptor-mediated signaling and photosynthesis play a role in determining the orientation of plant organs. TILLER ANGLE CONTROL 1 (TAC1) was recently shown to function in setting the orientation of lateral branches in diverse plant species, but the degree to which it plays a role in light-mediated phenotypes is unknown. Here, we demonstrated that TAC1 expression was light dependent, as expression was lost under continuous dark or far-red growth conditions, but did not drop to these low levels during a diurnal time course. Loss of TAC1 in the dark was gradual, and experiments with photoreceptor mutants indicated this was not dependent upon red/far-red or blue light signaling, but partially required the signaling integrator CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1). Overexpression of TAC1 partially prevented the narrowing of branch angles in the dark or under far-red light. Treatment with the carotenoid biosynthesis inhibitor norflurazon or the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) led to loss of TAC1 expression similar to dark or far-red conditions, but expression increased in response to the PSI inhibitor paraquat. Treatment of adult plants with norflurazon resulted in upward growth angle of branch tips. Our results indicate that TAC1 plays an important role in modulating plant architecture in response to photosynthetic signals.

Transcriptomic changes triggered by carotenoid biosynthesis inhibitors and role of Citrus sinensis phosphate transporter 4;2 (CsPHT4;2) in enhancing carotenoid accumulation.

Carotenoid accumulation and chromoplast development in orange were perturbed by carotenoid inhibitors, and candidate genes were identified via transcriptomic analysis. The role of CsPHT4;2 in enhancing carotenoid accumulation was revealed. Carotenoids are important plant pigments and their accumulation can be affected by biosynthesis inhibitors, but the genes involved were largely unknown. Here, application of norflurazon (NFZ), 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) and clomazone for 30days to in vitro cultured sweet orange juice vesicles caused over-accumulation of phytoene (over 1000-fold), lycopene (2.92μgg-1FW, none in control), and deficiency in total carotenoids (reduced to 22%), respectively. Increased carotenoids were associated with bigger chromoplasts with enlarged plastoglobules or a differently crystalline structure in NFZ, and CPTA-treated juice vesicles, respectively. Global transcriptomic changes following inhibitor treatments were profiled. Induced expression of 1-deoxy-D-xylulose 5-phosphate synthase 1 by CPTA, hydroxymethylbutenyl 4-diphosphate reductase by both NFZ and CPTA, and reduced expression of chromoplast-specific lycopene β-cyclase by CPTA, as well as several downstream genes by at least one of the three inhibitors were observed. Expression of fibrillin 11 (CsFBN11) was induced following both NFZ and CPTA treatments. Using weighted correlation network analysis, a plastid-type phosphate transporter 4;2 (CsPHT4;2) was identified as closely correlated with high-lycopene accumulation induced by CPTA. Transient over-expression of CsPHT4;2 significantly enhanced carotenoid accumulation over tenfold in 'Cara Cara' sweet orange juice vesicle-derived callus. The study provides a valuable overview of the underlying mechanisms for altered carotenoid accumulation and chromoplast development following carotenoid inhibitor treatments and sheds light on the relationship between carotenoid accumulation and chromoplast development.

Action mechanism of bleaching herbicide cyclopyrimorate, a novel homogentisate solanesyltransferase inhibitor.

The action mechanism of cyclopyrimorate, a novel herbicide for weed control in rice fields, was investigated. Cyclopyrimorate caused bleaching symptoms in Arabidopsis thaliana similar to those caused by existing carotenoid biosynthesis inhibitors, mesotrione and norflurazon. However, cyclopyrimorate treatment resulted in significant accumulation of homogentisate and a reduction in the level of plastoquinone. A metabolite of cyclopyrimorate, des-morpholinocarbonyl cyclopyrimorate (DMC), was detected in plants. These data suggested that cyclopyrimorate and/or DMC inhibit homogentisate solanesyltransferase (HST), a downstream enzyme of 4-hydroxyphenylpyruvate dioxygenase in the plastoquinone biosynthesis pathway. In vitro assays showed that A. thaliana HST was strongly inhibited by DMC and weakly by cyclopyrimorate, whereas other commercial bleaching herbicides did not inhibit HST. DMC derivatives showed a positive correlation between HST inhibition and in vivo bleaching activities. These results indicate that the target site of cyclopyrimorate and DMC is HST, a novel target site of commercial herbicides.

Perturbations in carotenoid and porphyrin status result in differential photooxidative stress signaling and antioxidant responses

We examined differential photooxidative stress signaling and antioxidant responses in rice plants treated with norflurazon (NF) and oxyfluorfen (OF), which are inhibitors of carotenoid and porphyrin biosynthesis, respectively. Plants treated with OF markedly increased levels of cellular leakage and malondialdehyde, compared with NF-treated plants, showing that OF plants suffered greater oxidative damage with respect to membrane integrity. The enhanced production of H2O2 in response to OF, but not NF, indicates the important role of H2O2 in activation of photooxidative stress signaling in OF plants. In response to NF and OF, the increased levels of free salicylic acid as well as maintenance of the redox ratio of ascorbate and glutathione pools to a certain level are considered to be crucial factors in the protection against photooxidation. Plants treated with OF greatly up-regulated catalase (CAT) activity and Cat transcript levels, compared with NF-treated plants. Interestingly, NF plants showed no noticeable increase in oxidative metabolism, although they did show considerable increases in ascorbate peroxidase (APX) and peroxidase activities and transcript levels of APX, as in OF plants. Our results suggest that perturbations in carotenoid and porphyrin status by NF and OF can be sensed by differential photooxidative stress signaling, such as that involving H2O2, redox state of ascorbate and glutathione, and salicylic acid, which may be responsible for at least part of the induction of ROS-scavenging enzymes.

In BPS1 Downregulated Roots, the BYPASS1 Signal Disrupts the Induction of Cortical Cell Divisions in Bean-Rhizobium Symbiosis.

BYPASS1 (BPS1), which is a well-conserved gene in plants, is required for normal root and shoot development. In the absence of BPS1 gene function, Arabidopsis overproduces a mobile signalling compound (the BPS1 signal) in roots, and this transmissible signal arrests shoot growth and causes abnormal root development. In addition to the shoot and root meristem activities, the legumes also possess transient meristematic activity in root cortical cells during Rhizobium symbiosis. We explored the role of Phaseolus vulgaris BPS1 during nodule primordium development using an RNA-interference (RNAi) silencing approach. Our results show that upon Rhizobium infection, the PvBPS1-RNAi transgenic roots failed to induce cortical cell divisions without affecting the rhizobia-induced root hair curling and infection thread formation. The transcript accumulation of early nodulin genes, cell cyclins, and cyclin-dependent kinase genes was affected in RNAi lines. Interestingly, the PvBPS1-RNAi root nodule phenotype was partially rescued by exogenous application of fluridone, a carotenoid biosynthesis inhibitor, which was used because the carotenoids are precursors of BPS1 signalling molecules. Furthermore, we show that the PvBPS1 promoter was active in the nodule primordia. Together, our data show that PvBPS1 plays a vital role in the induction of meristematic activity in root cortical cells and in the establishment of nodule primordia during Phaseolus-Rhizobium symbiosis.

Transcriptome analysis of Phelipanche aegyptiaca seed germination mechanisms stimulated by fluridone, TIS108, and GR24.

P. aegyptiaca is one of the most destructive root parasitic plants worldwide, causing serious damage to many crop species. Under natural conditions P. aegyptiaca seeds must be conditioned and then stimulated by host root exudates before germinating. However, preliminary experiments indicated that TIS108 (a triazole-type inhibitor of strigolactone) and fluridone (FL, an inhibitor of carotenoid-biosynthesis) both stimulated the germination of P. aegyptiaca seeds without a water preconditioning step (i.e. unconditioned seeds). The objective of this study was to use deep RNA sequencing to learn more about the mechanisms by which TIS108 and FL stimulate the germination of unconditioned P. aegyptiaca seeds. Deep RNA sequencing was performed to compare the mechanisms of germination in the following treatments: (i) unconditioned P. aegyptiaca seeds with no other treatment, (ii) unconditioned seeds treated with 100 mg/L TIS108, (iii) unconditioned seeds treated with 100 mg/L FL + 100 mg/L GA3, (iv) conditioned seeds treated with sterile water, and (v) conditioned seeds treated with 0.03 mg/L GR24. The de novo assembled transcriptome was used to analyze transcriptional dynamics during seed germination. The key gene categories involved in germination were also identified. The results showed that only 119 differentially expressed genes were identified in the conditioned treatment vs TIS108 treatment. This indicated that the vast majority of conditions for germination were met during the conditioning stage. Abscisic acid (ABA) and gibberellic acid (GA) played important roles during P. aegyptiaca germination. The common pathway of TIS108, FL+GA3, and GR24 in stimulating P. aegyptiaca germination was the simultaneous reduction in ABA concentrations and increase GA concentrations. These results could potentially aid the identification of more compounds that are capable of stimulating P. aegyptiaca germination. Some potential target sites of TIS108 were also identified in our transcriptome data. The results of this experiment suggest that TIS108 and FL+GA3 could be used to control P. aegyptiaca through suicidal germination.