The Interplay Between Water Limitation, Dhurrin, and Nitrate in the Low-Cyanogenic Sorghum Mutant adult cyanide deficient class 1.

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.

Label-free Raman hyperspectral imaging analysis localizes the cyanogenic glucoside dhurrin to the cytoplasm in sorghum cells.

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.

Counting the costs: nitrogen partitioning in Sorghum mutants.

Long-standing growth/defence theories state that the production of defence compounds come at a direct cost to primary metabolism when resources are limited. However, such trade-offs are inherently difficult to quantify. We compared the growth and nitrogen partitioning in wild type Sorghum bicolor (L.) Moench, which contains the cyanogenic glucoside dhurrin, with unique mutants that vary in dhurrin production. The totally cyanide deficient 1 (tcd1) mutants do not synthesise dhurrin at all whereas mutants from the adult cyanide deficient class 1 (acdc1) have decreasing concentrations as plants age. Sorghum lines were grown at three different concentrations of nitrogen. Growth, chemical analysis, physiological measurements and expression of key genes in biosynthesis and turnover were determined for leaves, stems and roots at four developmental stages. Nitrogen supply, ontogeny, tissue type and genotype were all important determinants of tissue nitrate and dhurrin concentration and turnover. The higher growth of acdc1 plants strongly supports a growth/defence trade-off. By contrast, tcd1 plants had slower growth early in development, suggesting that dhurrin synthesis and turnover may be beneficial for early seedling growth rather than being a cost. The relatively small trade-off between nitrate and dhurrin suggests these may be independently regulated.

Resilience of cassava (Manihot esculenta Crantz) to salinity: implications for food security in low-lying regions.

Rising sea levels are threatening agricultural production in coastal regions due to inundation and contamination of groundwater. The development of more salt-tolerant crops is essential. Cassava is an important staple, particularly among poor subsistence farmers. Its tolerance to drought and elevated temperatures make it highly suitable for meeting global food demands in the face of climate change, but its ability to tolerate salt is unknown. Cassava stores nitrogen in the form of cyanogenic glucosides and can cause cyanide poisoning unless correctly processed. Previous research demonstrated that cyanide levels are higher in droughted plants, possibly as a mechanism for increasing resilience to oxidative stress. We determined the tolerance of cassava to salt at two different stages of development, and tested the hypothesis that cyanide toxicity would be higher in salt-stressed plants. Cassava was grown at a range of concentrations of sodium chloride (NaCl) at two growth stages: tuber initiation and tuber expansion. Established plants were able to tolerate 100mM NaCl but in younger plants 40mM was sufficient to retard plant growth severely. Nutrient analysis showed that plants were only able to exclude sodium at low concentrations. The foliar cyanogenic glucoside concentration in young plants increased under moderate salinity stress but was lower in plants grown at high salt. Importantly, there was no significant change in the cyanogenic glucoside concentration in the tubers. We propose that the mechanisms for salinity tolerance are age dependent, and that this can be traced to the relative cost of leaves in young and old plants.

Interactive effects of temperature and drought on cassava growth and toxicity: implications for food security?

Cassava is an important dietary component for over 1 billion people, and its ability to yield under drought has led to it being promoted as an important crop for food security under climate change. Despite its known photosynthetic plasticity in response to temperature, little is known about how temperature affects plant toxicity or about interactions between temperature and drought, which is important because cassava tissues contain high levels of toxic cyanogenic glucosides, a major health and food safety concern. In a controlled glasshouse experiment, plants were grown at 2 daytime temperatures (23 °C and 34 °C), and either well-watered or subject to a 1 month drought prior to harvest at 6 months. The objective was to determine the separate and interactive effects of temperature and drought on growth and toxicity. Both temperature and drought affected cassava physiology and chemistry. While temperature alone drove differences in plant height and above-ground biomass, drought and temperature × drought interactions most affected tuber yield, as well as foliar and tuber chemistry, including C : N, nitrogen and cyanide potential (CNp; total cyanide released from cyanogenic glucosides). Conditions that most stimulated growth and yield (well-watered × high temperature) effected a reduction in tuber toxicity, whereas drought inhibited growth and yield, and was associated with increased foliar and tuber toxicity. The magnitude of drought effects on tuber yield and toxicity were greater at high temperature; thus, increases in tuber CNp were not merely a consequence of reduced tuber biomass. Findings confirm that cassava is adaptable to forecast temperature increases, particularly in areas of adequate or increasing rainfall; however, in regions forecast for increased incidence of drought, the effects of drought on both food quality (tuber toxicity) and yield are a greater threat to future food security and indicate an increasing necessity for processing of cassava to reduce toxicity.

Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

Focal plane array infrared imaging: a new way to analyse leaf tissue.

* Here, a new approach to macromolecular imaging of leaf tissue using a multichannel focal plane array (FPA) infrared detector was compared with the proven method of infrared mapping with a synchrotron source, using transverse sections of leaves from a species of Eucalyptus. * A new histological method was developed, ideally suited to infrared spectroscopic analysis of leaf tissue. Spatial resolution and the signal-to-noise ratio of the FPA imaging and synchrotron mapping methods were compared. * An area of tissue 350 microm(2) required approx. 8 h to map using the synchrotron technique and approx. 2 min to image using the FPA. The two methods produced similar infrared images, which differentiated all tissue types in the leaves according to their macromolecular chemistry. * The synchrotron and FPA methods produced similar results, with the synchrotron method having superior signal-to-noise ratio and potentially better spatial resolution, whereas the FPA method had the advantage in terms of data acquisition time, expense and ease of use. FPA imaging offers a convenient, laboratory-based approach to microscopic chemical imaging of leaves.