The Interplay Between Water Limitation, Dhurrin, and Nitrate in the Low-Cyanogenic Sorghum Mutant adult cyanide deficient class 1.
ABSTRACT: 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: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: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: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: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.
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: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:Members of the nitrilase 4 (NIT4) family of higher plants catalyze the conversion of beta-cyanoalanine to aspartic acid and asparagine, a key step in cyanide detoxification. Grasses (Poaceae) possess two different NIT4 homologs (NIT4A and NIT4B), but none of the recombinant Poaceae enzymes analyzed showed activity with beta-cyanoalanine, whereas protein extracts of the same plants clearly posses this activity. Sorghum bicolor contains three NIT4 isoforms SbNIT4A, SbNIT4B1, and SbNIT4B2. Individually, each isoform does not possess enzymatic activity whereas the heteromeric complexes SbNIT4A/B1 and SbNIT4A/B2 hydrolyze beta-cyanoalanine with high activity. In addition, the SbNIT4A/B2 complex accepts additional substrates, the best being 4-hydroxyphenylacetonitrile. Corresponding NIT4A and NIT4B isoforms from other Poaceae species can functionally complement the sorghum isoforms in these complexes. Site-specific mutagenesis of the active site cysteine residue demonstrates that hydrolysis of beta-cyanoalanine is catalyzed by the NIT4A isoform in both complexes whereas hydrolysis of 4-hydroxyphenylacetonitrile occurs at the NIT4B2 isoform. 4-Hydroxyphenylacetonitrile was shown to be an in vitro breakdown product of the cyanogenic glycoside dhurrin, a main constituent in S. bicolor. The results indicate that the SbNIT4A/B2 heterocomplex plays a key role in an endogenous turnover of dhurrin proceeding via 4-hydroxyphenylacetonitrile and thereby avoiding release of toxic hydrogen cyanide. The operation of this pathway would enable plants to use cyanogenic glycosides as transportable and remobilizable nitrogenous storage compounds. Through combinatorial biochemistry and neofunctionalizations, the small family of nitrilases has gained diverse biological functions in nitrile metabolism.
Project description:Chloride (Cl(-)) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a role as an unspecific cell osmoticum, no clear biological roles have been explicitly associated with Cl(-) when accumulated to macronutrient concentrations. To address this question, the glycophyte tobacco (Nicotiana tabacum L. var. Habana) has been treated with a basal nutrient solution supplemented with one of three salt combinations containing the same cationic balance: Cl(-)-based (CL), nitrate-based (N), and sulphate+phosphate-based (SP) treatments. Under non-saline conditions (up to 5 mM Cl(-)) and no water limitation, Cl(-) specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1-5 mM range, Cl(-) played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl(-) also altered water relations at the whole-plant level through reduction of plant transpiration. This was a consequence of a lower stomatal conductance, which resulted in lower water loss and greater photosynthetic and integrated water-use efficiency. In contrast to Cl(-), these effects were not observed for essential anionic macronutrients such as nitrate, sulphate, and phosphate. We propose that the abundant uptake and accumulation of Cl(-) responds to adaptive functions improving water homeostasis in higher plants.
Project description:In plants, the nitrate transporters, NRT1.1 and NRT2.1, are mainly responsible for nitrate uptake. Intriguingly, both nitrate transporters are located in a complementary manner in different cells layers of the mature root suggesting that their coordination should occur during nitrate uptake and plant growth. This hypothesis was examined on 5-d-old rape seedlings grown on agar medium supplemented with 1 or 5mM nitrate. Seedlings were treated with increasing potassium glutamate concentrations in order to uncouple the two nitrate transporters by inhibiting BnNRT2.1 expression and activity specifically. In both nitrate treatments, increasing the glutamate concentrations from 0.5 to 10mM induced a reduction in (15)NO 3(-) uptake and an inhibition of N assimilation. The decrease in (15)NO 3(-) uptake was caused by downregulation of BnNRT2.1 expression but surprisingly it was not compensated by the upregulation of BnNRT1.1. This created an unprecedented physiological situation where the effects of the nitrate signal on shoot growth were solely modulated by nitrate absorption. In these conditions, the osmotic water flow for volumetric shoot growth was mainly dependent on active nitrate transport and nitrate signaling. This behavior was confirmed by the allometric relationships found between changes in the root length with (15)N and water accumulation in the shoot. These findings demonstrate that the BnNRT2.1 transporter is essential for nitrate uptake and growth, and renew the question of the respective roles of the NRT2.1 and NRT1.1 transporters in nitrate uptake and sensing at the whole plant level.
Project description:Background and Aims: Understanding interactions between water and nitrate fluxes in response to nitrate availability and transpiration rate is crucial to select more efficient plants for the use of water and nitrate. Methods: Some of these interactions were investigated in intact Brassica napus plants by combining a non-destructive gravimetric device with 15NO3 - labeling. The set-up allowed high-resolution measurement of the effects of a cross-combination of two concentrations of KNO3 or KCl (0.5 and 5 mM) with two different rates of transpiration controlled by the relative humidity during a day-night cycle. Key Results: Results show that (1) high external nitrate concentrations increased root water uptake significantly whatever the transpiration rate, (2) nitrate translocation depended both on the rate of nitrate uptake and loading into xylem (3) dilution-concentration effect of nitrate in the xylem was mainly modulated by both external nitrate availability and transpiration rate, (4) dynamic changes in 15N translocation in the xylem modified shoot growth and capacitance, and (5) variations in tissue concentrations of NO3 - induced by the experimental conditions were balanced by changes in concentrations of chloride and sulfate ions. These effects were even more amplified under low transpiration condition and 0.5 mM external nitrate concentration. Conclusion: Taken together, these results highlight the fine and rapid adjustment of anion contents, nitrate and water flows to changes in transpiration rate and nitrate availability during a day-night cycle. The use of this non-invasive gravimetric device is therefore a powerful tool to assess candidates genes involved in nitrogen and water use efficiency.