Linamarin Levels Research Articles

Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava.

Cassava is the world's most essential food root crop, generating calories to millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of residual cyanogens results in cyanide poisoning due to conversion of the cyanogens to cyanide in the body. There is a need for acyanogenic cassava cultivars in order for it to become a consistently safe and acceptable food, and commercial crop. In recent years, the CRISPR/Cas system, has proven to be the most effective and successful genome editing tool for gene function studies and crop improvement. In this study, we performed targeted mutagenesis of the MeCYP79D1 gene in exon 3, using CRISPR/Cas9, via Agrobacterium-mediated transformation. The vector design resulted in knockout in cotyledon-stage somatic embryos regenerated under hygromycin selection. Eight plants were recovered and genotyped. DNA sequencing analysis revealed that the tested putative transgenic plants carried mutations within the MeCYP79D1 locus, with deletions and substitutions being reported upstream and downstream of the PAM sequence, respectively. The levels of linamarin and evolved cyanide present in the leaves of mecyp79d1 lines were reduced up to seven-fold. Nevertheless, the cassava linamarin and cyanide were not completely eliminated by the MeCYP79D1 knockout. Our results indicate that CRISPR/Cas9-mediated mutagenesis is as an alternative approach for development of cassava plants with lowered cyanide content.

Diurnal regulation of cyanogenic glucoside biosynthesis and endogenous turnover in cassava.

Cyanogenic glucosides are present in many plants, including eudicots, monocots, and ferns and function as defence compounds based on their ability to release hydrogen cyanide. In this study, the diurnal rhythm of cyanogenic glucoside content and of transcripts and enzymes involved in their biosynthesis was monitored in cassava plants grown in a glasshouse under natural light conditions. Transcripts of CYP79D1, CYP79D2, CYP71E7/11, and UGT85K5 were at minimal levels around 9 p.m., increased during the night and decreased following onset of early morning light. Transcripts of UGT85K4 and HNL10 showed more subtle variations with a maximum reached in the afternoon. Western blots showed that the protein levels of CYP71E7/11 and UGT85K4/5 decreased during the light period to a near absence around 4 p.m. and then recovered during the dark period. Transcript and protein levels of linamarase were stable throughout the 24‐hr cycle. The linamarin content increased during the dark period. In the light period, spikes in the incoming solar radiation were found to result in concomitantly reduced linamarin levels. In silico studies of the promoter regions of the biosynthetic genes revealed a high frequency of light, abiotic stress, and development‐related transcription factor binding motifs. The synthesis and endogenous turnover of linamarin are controlled both at the transcript and protein levels. The observed endogenous turnover of linamarin in the light period may offer a source of reduced nitrogen to balance photosynthetic carbon fixation. The rapid decrease in linamarin content following light spikes suggests an additional function of linamarin as a ROS scavenger.

Genetic, morphological and cyanogen content evaluation of a new collection of Caribbean Lima bean (Phaseolus lunatus L.) landraces

Lima bean is a species cultivated broadly in the Americas and has been cultivated in the Caribbean for at least 500 years. In order to determine the genetic structure and diversity of Lima bean from the Caribbean, 50 landraces from Haiti, the Dominican Republic and Puerto Rico were collected and analyzed using 24 SSR markers. All landraces in this Caribbean collection were found to be of Middle-American descent. The genetic diversity (HE) was highest in the landraces from Puerto Rico and lowest in Haitian landraces. The observed proportion of heterozygotes (HO) was higher in the Haitian landraces and lowest in Puerto Rican landraces. Unweighted pair-group method with arithmetic averaging analysis showed that the landraces clustered into 3 clusters with all Haitian landraces grouping in one cluster. We also assessed the agro-morphological characteristics of the collection as well as the content of cyanogenic glucoside, linamarin, in leaves and dry seeds. Lima bean, which is a model crop for indirect plant defenses against herbivory, also possess linamarin as a source of direct plant defense. Upon tissue damage, linamarin is converted to toxic hydrogen cyanide. In our collection 44.6 % of the landraces had average seed HCN content ≤200 ppm which is the permitted level for Lima bean seed in the US. Our results also identified the landraces in this collection which have high linamarin levels in the leaves while having low levels in the seeds. Such landraces have the desirable combination of traits and will be the focus of our future plans for agronomic trait improvement though breeding.

PENGOLAHAN BIJI KARET SEBAGAI BAHAN BAKU PEMBUATAN MINYAK PANGAN (EDIBLE OIL)

<p>Abstract: Rubber (Hevea Brasiliensa)is one of Indonesia's largest plantation commodities is not<br />optimal utilization. Rubber seeds have a high vegetable oil content of 45.63%, so the potential to<br />be used as edible oil. However, toxic linamarin contained in the seeds of an obstacle in the<br />utilization of rubber, because if hydrolyzed to form hydrogen cyanide (HCN), which if consumed<br />in large amounts can cause dizziness and fainting. Cyanide is soluble and evaporates. This study<br />aims to provide early treatment for rubber seed, in order to reduce levels of toxic linamarin. This<br />research is still exploratory, as a preliminary study for rubber seed oil to prepare adequate food<br />consumption. Linamarin removal process is done by soaking and boiling with variable immersion<br />1-3 days old, and the old variable boiling 1-2 hours. Rubber seed which has undergone initial<br />treatment further analyzed by the method of Volhard Argentometri way to determine levels of<br />linamarin contained therein. Furthermore, the oil extraction process is carried out using the<br />solvent n-hexane followed by degumming process and analysis of petroleum oil based food<br />quality standards (SNI 01-3741-2002)with parameters and acid value metal content. From the<br />research that has been done, the result that the greatest decline linamarin levels achieved at 1<br />day of immersion time and 1.5 hours of boiling time, ie by 39%. The results of the metal analysis<br />showed that rubber seed oil rubber seed oil oils do not meet the quality standards of food because<br />it contains a metal content of 1.78 ppm Cu (oil food safe limit of 1 ppm)that required further<br />treatment to reduce levels of Cu, one how the adsorption process using a specific adsorber.<br />Keywords: Rubber, Linamarin, Edible Oil.</p>

Isolation and screening of microorganisms from a gari fermentation process for starter culture development

Cassava ( Manihot esculenta Crantz ), is used for the production of a variety of West African foods and ranks fourth in the list of major crops in developing countries after rice, wheat and maize. Gari is one of the most popular foods produced from cassava. Cassava may contain high levels of linamarin, a cyanogenic glucoside, which in its natural state is toxic to man. Therefore, some processing methods that can enhance the detoxification of cassava and lead to the improvement of the quality and hygienic safety of the food are vitally important for less toxic products to be obtained. Quality, safety and acceptability of traditional fermented foods may be improved through the use of starter cultures. There has been a trend recently to isolate wild-type strains from traditional products for use as starter cultures in food fermentation. A total of 74 bacterial strains and 21 yeast strains were isolated from a cassava mash fermentation process in a rural village in Benin, West Africa. These strains were assessed, together with 26 strains isolated at the Council for Scientific and Industrial Research (CSIR) from cassava samples sent from Benin previously, for phenotypic and technological properties. 24 presumptive lactic acid bacteria (LAB) were selected for further phenotypic, genotypic and technological characterization. Key words : Lactic acid bacteria, gari, cassava, fermentation.

Overexpression of Hydroxynitrile Lyase in Cassava Roots Elevates Protein and Free Amino Acids while Reducing Residual Cyanogen Levels

Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2–20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

Engineering cyanogen synthesis and turnover in cassava (Manihot esculenta)

Cassava is the major root crop for a quarter billion subsistence farmers in sub-Saharan Africa. It is valued for its ability to grow in adverse environments and the food security it provides. Cassava contains potentially toxic levels of cyanogenic glycosides (linamarin) which protect the plant from herbivory and theft. The cyanogens, including linamarin and its deglycosylated product, acetone cyanohydrin, can be efficiently removed from the root by various processing procedures. Short-cuts in processing, which may occur during famines, can result in only partial removal of cyanogens. Residual cyanogens in cassava foods may cause neurological disorders or paralysis, particularly in nutritionally compromised individuals. To address this problem and to further understand the function of cyanogenic glycosides in cassava, we have generated transgenic cassava in which cyanogenic glycoside synthesis has been selectively inhibited in leaves and roots by antisense expression of CYP79D1/D2 gene fragments. The CYP79D1/D2 genes encode two highly similar cytochrome P450s that catalyze the first-dedicated step in cyanogenic glycoside synthesis. Transgenic plants in which the expression of these genes was selectively inhibited in leaves had substantially reduced (60- 94% reduction) linamarin leaf levels. Surprisingly, these plants also had a greater than a 99% reduction in root linamarin content. In contrast, transgenic plants in which the CYP79D1/D2 transcripts were reduced to non-detectable levels in roots had normal root linamarin levels. These results demonstrate that linamarin synthesized in leaves is transported to the roots and accounts for nearly all of the root linamarin content. Importantly, transgenic plants having reduced leaf and root linamarin content were unable to grow in the absence of reduced nitrogen (NH3) . Cassava roots have previously been demonstrated to have an active cyanide assimilation pathway leading to the synthesis of amino acids. We propose that cyanide derived from linamarin is a major source of reduced nitrogen for cassava root protein synthesis. Disruption of linamarin transport from leaves in CYP79D1/D2 anti-sense plants prevents the growth of cassava roots in the absence of an alternate source of reduced nitrogen. An alternative strategy for reducing cyanogen toxicity in cassava foods is to accelerate cyanogenesis and cyanide volatilization during food processing. To achieve this objective, we have expressed the leaf-specific enzyme hydroxynitrile lyase (HNL) in roots. HNL catalyzes the breakdown of acetone cyanohydrin to cyanide. Expression of HNL in roots accelerated cyanogenesis by more than three-fold substantially reducing the accumulation of acetone cyanohydrin during processing relative to wild-type roots.

Low dietary cyanogen exposure from frequent consumption of potentially toxic cassava in Malawi

In a cassava-growing area in Malawi, where roots are processed by soaking and water is available throughout the year, we interviewed 176 women farmers regarding their preferences for cassava cultivars and frequency of cassava consumption. Dietary cyanogen exposure was estimated from urinary levels of linamarin, the cyanogenic glycoside in cassava, and urinary thiocyanate, the main cyanide metabolite. Protection against unplanned harvest by family members, theft and animal spoilage were stated to be very important reasons for growing bitter cassava cultivars by 91%, 90% and 74% of the women, respectively. The mean (±SD) number of cultivars grown by each woman was 4.6 (±2.4). The correlation between mean taste and mean danger scores for the 25 most grown cultivars was strong (r > 0.98). The scoring indicated that cultivars belonged to two distinct groups, eight to a group referred to as 'cool' and 17 to a group termed 'bitter'. The dumpling-like porridge (kondowole) made from cassava flour from bitter roots was eaten twice daily by 51% and at least weekly by 81%. The mean (±SEM) urinary linamarin was 14 (±1) micromol/L and thiocyanate was 50 (±4) micromol/L, less than a tenth of levels reported from populations eating insufficiently processed bitter cassava roots, and in the same range as in a non-smoking Swedish reference population. We conclude that cyanogenesis is a preferred characteristic of cassava by the studied farmers because it enhances food security. The availability of water and their knowledge about toxicity and processing enables these women farmers to provide a safe staple food from bitter cassava roots.

Cyanogenic Glucosides In Linum Usitatissimum

Cyanogenic glucosides were quantified in different organs of oil flax ( Linum usitatissimum cv LCSD 200) plants at different stages of development. Monoglucosides (linamarin and lotaustralin) and diglucosides (linustatin and neolinustatin) appeared in developing embryos soon after anthesis, but mature seeds accumulated only diglucosides. Monoglucosides appeared again in germinating seeds and, in young seedlings, they were the only class of cyanogens. High levels of linamarin and lotaustralin were found in leaves throughout the vegetation period, but the highest amounts were in flowers. In contrast, these glucosides occurred in relatively small amounts in roots and in stems. The possible physiological roles of the changes are discussed.

Low cyanide exposure from consumption of cassava in Dar es Salaam, Tanzania.

The extent of cyanide exposure from cassava consumption was studied in low income suburbs of Dar es Salaam, Tanzania. Mean cyanogen levels in sun-dried root pieces called makopa was 9.4 (range 0-79) mg HCN equivalents kg(-1) dry weight. The mean glucoside and hydrogen cyanide levels were 6.4 and 3.2 mg HCN equivalents kg(-1) dry weight, respectively, while cyanohydrins were lower with a mean of 2.0 (range 0-27) equivalents kg(-1) dry weight. Food frequency interviews with 193 schoolchildren revealed that 13% of the children consumed cassava stiff porridge in the previous week. Fried cassava pieces were consumed by 82% and boiled cassava pieces by 49% of the children. The urinary thiocyanate in these children was 36 +/- 3 (mean +/- SEM) micromol l(-1) and mean urinary linamarin level was 18 +/- 1 micromol l(-1), indicating low cyanide exposure. Multiple regression analysis revealed a positive correlation between urinary thiocyanate and consumption of boiled cassava pieces as well as between urinary linamarin levels and daily intake of fried cassava pieces.

Cyanogen removal from cassava roots during sun-drying

Linamarin levels in sun-drying cassava root pieces showed an exponential decrease, parallel with the moisture loss, and stabilized when moisture levels reached about 15%. Linamarin degradation in thin root segments was significantly slower and less complete than in thick ones. Disinfected longitudinal root halves, oven-dried at 40 °C for 24 h, had significantly higher residual linamarin levels than the matched ones subjected to humid incubation at 25 °C. Interrupting for one or two days the sun-drying of peeled roots resulted in significantly lower residual levels of linamarin and of cyanohydrins plus HCN. Linamarin degradation was greater when the interruption was earlier or longer. The rate of dehydration influences linamarin degradation negatively — mechanisms are discussed. Cyanohydrin removal was completed by prolonged drying. Sun-drying is not very effective in linamarin removal, and speeding up the drying process, e.g. by reducing the size of the pieces, aggravates this. There is a potential for increasing the effectiveness of cyanogen removal by reducing the initial drying rate, followed by thorough final drying.

Microdiffusion method with solid state detection of cyanogenic glycosides from cassava in human urine

A method for quantitative determination of cyanogenic glycosides in human urine is described. It is based on enzymatic cleavage of the glycosides, microdiffusion of the hydrogen cyanide formed, solid state detection by colour formation on a picrate-impregnated sheet, and subsequent rating of the coloured spot by the absorption of transmitted light at 540 nm with a thin-layer (TLC) densitometer. The method has been tested using normal as well as pathological urines containing glucose, protein, leucocytes, blood and bacteria. The method allows quantification of urinary linamarin above 70 μmol/litre, in 40 μl urine. In Mozambican subjects consuming insufficiently processed cassava the mean urinary linamarin levels were 211 μmol/litre, indicating for the first time that substantial amounts of the main cyanogenic glycoside in cassava may be absorbed from the human gut and excreted intact in the urine.

Purification, Characterization, and Localization of Linamarase in Cassava

We have purified cassava (Manihot esculenta) linamarase to apparent homogeneity using a simplified extraction procedure using low pH phosphate buffer. Three isozymes of cassava linamarase were identified in leaves based on differences in isoelectric point. The enzyme is capable of hydrolyzing a number of beta-glycosides in addition to linamarin. The enzyme is unusually stable and has a temperature optimum of 55 degrees C. Immunogold labeling studies indicate that linamarase is localized in the cell walls of cassava leaf tissue. Since linamarin must cross the cell wall following synthesis in the leaf for transport to the root, it is likely that linamarin must cross the cell wall in a nonhydrolyzable form, possibly as the diglucoside, linustatin. In addition, we have quantified the levels of linamarin and linamarase activity in leaves of cassava varieties which differ in the linamarin content of their roots. We observed no substantial differences in the steady state linamarin content or linamarase activity of leaves from high or low (root) cyanogenic varieties. These results indicate that the steady state levels of linamarin and linamarase in leaves of high and low cyanogenic varieties are not correlated with the varietal differences in the steady state levels of linamarin in roots.