Down-Regulation of CYP79A1 Gene Through Antisense Approach Reduced the Cyanogenic Glycoside Dhurrin in [Sorghum bicolor (L.) Moench] to Improve Fodder Quality.
ABSTRACT: A major limitation for the utilization of sorghum forage is the production of the cyanogenic glycoside dhurrin in its leaves and stem that may cause the death of cattle feeding on it at the pre-flowering stage. Therefore, we attempted to develop transgenic sorghum plants with reduced levels of hydrogen cyanide (HCN) by antisense mediated down-regulation of the expression of cytochrome P450 CYP79A1, the key enzyme of the dhurrin biosynthesis pathway. CYP79A1 cDNA was isolated and cloned in antisense orientation, driven by rice Act1 promoter. Shoot meristem explants of sorghum cultivar CSV 15 were transformed by the particle bombardment method and 27 transgenics showing the integration of transgene were developed. The biochemical assay for HCN in the transgenic sorghum plants confirmed significantly reduced HCN levels in transgenic plants and their progenies. The HCN content in the transgenics varied from 5.1 to 149.8 ?g/g compared to 192.08 ?g/g in the non-transformed control on dry weight basis. Progenies with reduced HCN content were advanced after each generation till T3. In T3 generation, progenies of two promising events were tested which produced highly reduced levels of HCN (mean of 62.9 and 76.2 ?g/g, against the control mean of 221.4 ?g/g). The reduction in the HCN levels of transgenics confirmed the usefulness of this approach for reducing HCN levels in forage sorghum plants. The study effectively demonstrated that the antisense CYP79A1 gene deployment was effective in producing sorghum plants with lower HCN content which are safer for cattle to feed on.
Project description:Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.
Project description:Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.
Project description:Sorghum (Sorghum bicolor (L.)) Moench is an important food for humans and feed for livestock. Sorghum contains dhurrin which can be degraded into toxic hydrogen cyanide. Here, we report the expression patterns of 14 candidate genes related to dhurrin ((S)-4-Hydroxymandelnitrile-?-D-glucopyranoside) metabolism and the effects of the gene expression on specific metabolite content in selected sorghum accessions. Dhurrin-related metabolism is vigorous in the early stages of development of sorghum. The dhurrin contents of most accessions tested were in the range of approximately 6-22 ?g mg-1 fresh leaf tissue throughout growth. The p-hydroxybenzaldehyde (pHB) contents were high at seedling stages, but almost nonexistent at adult stages. The contents of p-hydroxyphenylacetic acid (pHPAAc) were relatively low throughout growth compared to those of dhurrin or pHB. Generally, the expression of the candidate genes was higher at seedling stage than at other stages and decreased gradually as plants grew. In addition, we identified significant SNPs, and six of them were potentially associated with non-synonymous changes in CAS1. Our results may provide the basis for choosing breeding materials to regulate cyanide contents in sorghum varieties to prevent HCN toxicity of livestock or to promote drought tolerance or pathogen resistance.
Project description:Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products.
Project description:Sorghum bicolor (L.) Moench produces the nitrogen-containing natural product dhurrin that provides chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide gas. Drought can increase dhurrin in shoot tissues to concentrations toxic to livestock. As dhurrin is also a remobilizable store of reduced nitrogen and plays a role in stress mitigation, reductions in dhurrin may come at a cost to plant growth and stress tolerance. Here, we investigated the response to an extended period of water limitation in a unique EMS-mutant adult cyanide deficient class 1 (acdc1) that has a low dhurrin content in the leaves of mature plants. A mutant sibling line was included to assess the impact of unknown background mutations. Plants were grown under three watering regimes using a gravimetric platform, with growth parameters and dhurrin and nitrate concentrations assessed over four successive harvests. Tissue type was an important determinant of dhurrin and nitrate concentrations, with the response to water limitation differing between above and below ground tissues. Water limitation increased dhurrin concentration in the acdc1 shoots to the same extent as in wild-type plants and no growth advantage or disadvantage between the lines was observed. Lower dhurrin concentrations in the acdc1 leaf tissue when fully watered correlated with an increase in nitrate content in the shoot and roots of the mutant. In targeted breeding efforts to down-regulate dhurrin concentration, parallel effects on the level of stored nitrates should be considered in all vegetative tissues of this important forage crop to avoid potential toxic effects.
Project description:Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, "nanodiscs", and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80?±?5?mV and 72?±?5?mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90?±?5?mV and -300?±?10?mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s(-1). POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products.
Project description:BACKGROUND:The important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed. RESULTS:In this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. Dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. During the subsequent maturation period, the dhurrin content was turned over, resulting in only negligible residual dhurrin amounts in the mature grain. Dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. Despite the accumulation of dhurrin, the grains were acyanogenic as demonstrated by the lack of hydrogen cyanide release from macerated grain tissue and by the absence of transcripts encoding dhurrinases. With the missing activity of dhurrinases, the decrease in dhurrin content in the course of grain maturation represents the operation of hitherto uncharacterized endogenous dhurrin turnover pathways. Evidence for the operation of two such pathways was obtained by metabolite profiling and time-resolved transcriptome analysis. By combining cluster- and phylogenetic analyses with the metabolite profiling, potential gene candidates of glutathione S-transferases, nitrilases and glycosyl transferases involved in these pathways were identified. The absence of dhurrin in the mature grain was replaced by a high content of proanthocyanidins. Cluster- and phylogenetic analyses coupled with metabolite profiling, identified gene candidates involved in proanthocyanidin biosynthesis in sorghum. CONCLUSIONS:The results presented in this article reveal the existence of two endogenous dhurrin turnover pathways in sorghum, identify genes putatively involved in these transformations and show that dhurrin in addition to its insect deterrent properties may serve as a storage form of reduced nitrogen. In the course of sorghum grain maturation, proanthocyanidins replace dhurrin as a defense compound. The lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study CNglc synthesis as well as endogenous turnover.
Project description:Localisation of metabolites in sorghum coleoptiles using Raman hyperspectral imaging analysis was compared in wild type plants and mutants that lack cyanogenic glucosides. This novel method allows high spatial resolution in situ localization by detecting functional groups associated with cyanogenic glucosides using vibrational spectroscopy. Raman hyperspectral imaging revealed that dhurrin was found mainly surrounding epidermal, cortical and vascular tissue, with the greatest amount in cortical tissue. Numerous "hotspots" demonstrated dhurrin to be located within both cell walls and cytoplasm adpressed towards the plasmamembrane and not in the vacuole as previously reported. The high concentration of dhurrin in the outer cortical and epidermal cell layers is consistent with its role in defence against herbivory. This demonstrates the ability of Raman hyperspectral imaging to locate cyanogenic glucosides in intact tissues, avoiding possible perturbations and imprecision that may accompany methods that rely on bulk tissue extraction methods, such as protoplast isolation.
Project description:A Na+/H+ antiporter-like protein (NHXLP) was isolated from Sorghum bicolor L. (SbNHXLP) and validated by overexpressing in tomato for salt tolerance. Homozygous T2 transgenic lines when evaluated for salt tolerance, accumulated low Na+ and displayed enhanced salt tolerance compared to wild-type plants (WT). This is consistent with the amiloride binding assay of the protein. Transgenics exhibited higher accumulation of proline, K+, Ca2+, improved cambial conductivity, higher PSII, and antioxidative enzyme activities than WT. Fluorescence imaging results revealed lower Na+ and higher Ca2+ levels in transgenic roots. Co-immunoprecipitation experiments demonstrate that SbNHXLP interacts with a Solanum lycopersicum cation proton antiporter protein2 (SlCHX2). qRT-PCR results showed upregulation of SbNHXLP and SlCHX2 upon treatment with 200 mM NaCl and 100 mM potassium nitrate. SlCHX2 is known to be involved in K+ acquisition, and the interaction between these two proteins might help to accumulate more K+ ions, and thus maintain ion homeostasis. These results strongly suggest that plasma membrane bound SbNHXLP involves in Na+ exclusion, maintains ion homeostasis in transgenics in comparison with WT and alleviates NaCl stress.
Project description:Cyanogenic glycosides are defense compounds found in a wide range of plant species, including many crops. We demonstrate that the cyanogenic glucoside dhurrin, naturally found in sorghum, can be produced at high titers in Saccharomyces cerevisiae, constituting the first report of cyanogenic glycoside production in a microbe. Genetic modifications to increase the supply of the dhurrin precursor tyrosine enabled dhurrin production in excess of 80?mg/L. The dhurrin-producing yeast strain was used as a chassis to investigate previously uncharacterized enzymes identified close to the biosynthetic gene cluster containing the dhurrin pathway enzymes. This work shows the potential of heterologous expression in yeast to facilitate investigations of plant cyanogenic glycoside pathways.