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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.

Arbila beans contains cyanogenic compounds in the form of linamarin (cyanoglucosides), acetone cyanohydrin, and free cyanide, all together constitute total cyanide content. The objective of this study was to compare the cyanide content in arbila beans analyzed by picrate and acid hydrolysis methods. Picrate method measures total cyanide only. Cyanide content was identified by using a picrate paper, which turned into yellow. The absorbance was measured by a spectrophotometer at 510 nm. Acid hydrolysis method measures cyanide in arbila beans in the form of linamarin, acetone cyanohydrin, and free cyanide. Linamarin wa hydrolyzed in H 3 PO 4 solution. Estimated cyanide levels was measured by using a colorimetric procedure. Data was analyzed using Independent sample t-test (SPSS v.16). The results showed that, there was no difference in the total level of cyanide in both methods. Total cyanide measured by picrate and acid hydrolysis method was 2705.17 ppm and 2693.29 ppm, respectively. In addition, the three forms of cyanide content were as follows: linamarin 926.22 ppm, cyanohydrin 556.01 ppm, and free cyanide 1211.06 ppm. Based on the results, both methods can be used for total cyanide analysis. To determine the form of cyanide other than total cyanide, it is recommended to use the acid hydrolysis method.

The basic helix-loop-helix (bHLH) proteins are a large superfamily of transcription factors, and play a central role in a wide range of metabolic, physiological, and developmental processes in higher organisms. However, systematic investigation of bHLH gene family in cassava ( Manihot esculenta Crantz) has not been reported. In the present study, we performed a genome-wide survey and identified 148 MebHLH s genes were unevenly harbored in 18 chromosomes. Through phylogenetic analyses along with Arabidopsis counterparts, these MebHLHs genes were divided into 19 groups, and each gene contains a similar structure and conserved motifs. Moreover, many cis -acting regulatory elements related to various defense and stress responses showed in MebHLH genes. Interestingly, transcriptome data analyses unveiled 117 MebHLH genes during postharvest physiological deterioration (PPD) process of cassava tuberous roots, while 65 MebHLH genes showed significantly change. Meanwhile, the relative quantitative analysis of 15 MebHLH genes demonstrated that they were sensitive to PPD, suggesting they may involve in PPD process regulation. Cyanogenic glucosides (CGs) biosynthesis during PPD process was increased, silencing of MebHLH72 and MebHLH114 showed that linamarin content was significantly decreased in the leaves. To summarize, the genome-wide identification and expression profiling of MebHLH candidates pave a new avenue for uderstanding their function in PPD and CGs biosynthesis, which will accelerate the improvement of PPD tolerance and decrease CGs content in cassava tuberous roots.

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 100 mM NaCl but in younger plants 40 mM 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.

Cassava leaves have significant nutritional potential, but their high content of cyanogenic glycosides, which are synthesized as a defense mechanism and release hydrogen cyanide (HCN), poses a challenge using leaves as a potential food. This study evaluated the variation of HCN content in cassava leaves from seven and five cultivars at two stages after planting, respectively (seven and nine months). HCN levels ranged from 1800 to 2761 mg kg-1 (ppm) at seven months of age, with significant biological variation observed within cultivars. A reduction in HCN content was noted at nine months of age, with a maximum decrease of 74 % in one cultivar. Additionally, treatments involving crushing leaves followed boiling with NaHCO3 resulted in 90 % HCN reduction. This research highlights the importance of understanding HCN variability across cultivars and plant ages and suggests practical methods to reduce HCN content in leaves, making them a potential safer alternative nutritional source. Las hojas de yuca presentan un potencial nutricional significativo; sin embargo, su alto contenido en glucósidos cianogénicos, los cuales son sintetizados como un mecanismo de defensa de la planta, liberan cianuro de hidrógeno (HCN). Esta situación plantea un desafío para su uso como fuente alimentaria potencial. Este estudio evaluó la variación en el contenido de HCN en hojas de yuca de siete y cinco cultivares en dos etapas de desarrollo post-siembra, respectivamente (siete y nueve meses). Los niveles de HCN variaron entre 1800 y 2761 mg kg⁻¹ (ppm) a los siete meses, observándose una variación biológica significativa dentro de los cultivares. Se observó una reducción en el contenido de HCN a los nueve meses, con una disminución máxima del 74 % en uno de los cultivares. Además, los tratamientos que involucraron triturado de las hojas seguido de ebullición con NaHCO₃ resultaron en una reducción del 90 % de HCN. Esta investigación resalta la importancia de comprender la variabilidad del HCN entre cultivares y edades de las plantas, y sugiere métodos prácticos para reducir su contenido, lo que podría convertir a las hojas de yuca en una fuente nutricional alternativa más segura.

Secondary plant metabolites (SPMs) can influence the reduction of enteric methane (CH4) emissions in ruminants. This study aimed to evaluate the effects of supplementing diet with cyanogenic glucoside linamarin (LIN), essential oil eugenol (EU), and their combination (LIN+EU) on methanogenic microorganisms, CH4 production, and rumen fermentation parameters in vitro. The basal diet (BD) (alfalfa [Medicago sativa L.] hay and oat [Avena sativa L.] grain in a 3:1 ratio) was supplemented with LIN, EU, and LIN+EU mixtures and placed in amber bottles. The experimental treatments were: T1, BD (control); T2, BD-LIN (20 mg L-1); T3, BD-LIN (40 mg L-1); T4, BD-EU (400 mg L-1); T5, BD-LIN+EU (20 mg L-1 + 400 mg L-1); and T6, BD-LIN+EU (40 mg L-1 + 400 mg L-1). The treatments were inoculated with rumen fluid and incubated for 6, 12, and 24 h to assess abundance of total bacteria (TB), methanogenic archaea, and ruminal fermentation parameters. The CH4 production was significantly reduced (p < 0.001) with inclusion of LIN, EU, and LIN+EU, particularly at 12 and 24 h incubation. However, at these same time points, in vitro DM disappearance (IVDMD) was also reduced (p < 0.001), except when LIN was included alone, highlighting its advantage in this variable. Rumen pH remained relatively stable at 6 and 12 h but decreased (p < 0.001) at 24 h with LIN. The pH values ranged between 6.65 (minimum) and 6.85 (maximum), which are optimal for microbial activity. Total bacterial abundance (TB) was not affected (p > 0.05) by treatments, but methanogenic archaea abundance was significantly reduced (p < 0.001) at 24 h with the LIN+EU mixtures (T5 and T6), coinciding with the highest CH4 reductions of 36.9% and 56.7%, respectively. At 24 h, IVDMD with LIN alone was 51.83%, whereas EU alone resulted in 46.48%. In conclusion, LIN+EU mixtures T5 and T6 had a synergistic effect, effectively reducing CH4 emissions, demonstrating the combined impact of different SPMs mechanisms of action.

The high content of cyanogenic glycosides (CG) in cassava tubers affects food safety. CG are involved in the plant growth and development and protect cassava leaves from herbivorous predators. However, the regulatory mechanism of CG biosynthesis remains poorly understood. Here, yeast one-hybrid assays were performed using a mixed cDNA library of cassava tubers and leaves as prey and the promoter of MeCYP79D2 as bait. MeCYP79D2, a cytochrome P450 protein, is the rate-limiting enzyme for CG synthesis in cassava. From this information, a candidate regulator of MeCYP79D2 was selected and identified as transcription factor MePHD1.2. MePHD1.2, located in the nucleus and exhibiting an inhibitory transcription activity directly bound to an AT-rich motif in the promoter of MeCYP79D2. In cassava, the transcriptional activity of MeCYP79D2 was considerably enhanced in mephd1.2 mutant lines leading to increased linamarin and lotaustralin contents. Deletion of MePHD1.2 promoted the production of CGs in cassava and decreased transcription inhibition on MeCYP79D2, exposing a novel regulatory module governing biosynthesis of CGs.

Mass spectrometry based imaging is a powerful tool to investigate the spatial distribution of a broad range of metabolites across a variety of sample types. The recent developments in instrumentation and computing capabilities have increased the mass range, sensitivity and resolution and rendered sample preparation the limiting step for further improvements. Sample preparation involves sectioning and mounting followed by selection and application of matrix. In plant tissues, labile small molecules and specialized metabolites are subject to degradation upon mechanical disruption of plant tissues. In this study, the benefits of cryo-sectioning, stabilization of fragile tissues and optimal application of the matrix to improve the results from MALDI mass spectrometry imaging (MSI) is investigated with hydroxynitrile glucosides as the main experimental system. Denatured albumin proved an excellent agent for stabilizing fragile tissues such as leaves. In stem cross sections of , maintaining the samples frozen throughout the sectioning process and preparation of the samples by freeze drying enhanced the obtained signal intensity by twofold to fourfold. Deposition of the matrix by sublimation improved the spatial information obtained compared to spray. The imaging demonstrated that the cyanogenic glucosides (CNglcs) were localized in the vascular tissues in old stems of and in the periderm and vascular tissues of tubers. In MALDI mass spectrometry, the imaged compounds are solely identified by their ratio. MG20 and the mutant that is devoid of hydroxynitrile glucosides were used as negative controls to verify the assignment of the observed masses to linamarin, lotaustralin, and linamarin acid.

In 2009, Food Standards Australia New Zealand set a total cyanide content limit of 10 ppm for ready-to-eat cassava products to address food safety concerns about cyanogenic glucosides in cassava. This study surveys a range of cassava food products available in Melbourne, Australia, ten years after the implementation of these regulations. Of all the products tested, the mean cyanide content was greatest in ready-to-eat cassava chips (48.4 ppm), although imported ready-to-eat products had a higher mean cyanide content (95.9 ppm) than those manufactured in Australia (1.0 ppm). Cyanide was detected in frozen cassava products (grated mean = 12.9 ppm; whole root mean = 19.8 ppm), but was significantly reduced through processing according to packet instructions in both product types. Three methods were used to quantify total cyanide content: the evolved cyanide method, the picrate absorbance method and the picrate chart method, with satisfactory agreement between methods. The picrate absorbance and chart methods reported mean cyanide contents 13.7 ppm and 23.1 ppm higher, respectively, than the evolved cyanide method. Our results reaffirm the need for the ongoing testing of cassava food products, especially ready-to-eat products whose cyanide content will not be reduced before consumption.

Cyanide is a toxic substance found in several tubers such as cassava ( Manihot esculenta ), wild yam ( Dioscorea hispida Dennts), some cerealia and legumes. In the plants, it can be in the form of cyanogenic glycosides, acetone cyanohydrin, and hydrogen cyanide (HCN). Cyanogenic glycosides such as linamarin and lotaustralin belong to the product of secondary metabolism. The characteristic of cyanogenic glycosides is intermediately polar, water-soluble, and often accumulated in the vacuoles of plant cells. Acetone cyanohydrins are intermediates product from cyanogenic glycosides and cyanide acid in plant tissues. HCN, hydrogen cyanide, is a volatile and water-soluble compound. The toxic effect of cyanide in humans is inactivation of cytochrome oxidase, respiratory disorders, sore throat, dizziness, limpness, convulsions, and lethal effects. Cyanide can be removed by several processes before consumption. Methods such as peeling, washing, heating, drying, fermenting and chemical treatment are used to remove or reduce cyanide. The treatment can damage the structure of the cell and hydrolyzed the cyanogenic glycosides to acetone cyanohydrin and glucose by endogenous enzyme like linamarase. A second enzyme, hydroxynitril lyase, can dissociate acetone cyanohydrins to HCN, ketone and aldehyde compound. The maximum safe level for total cyanide in food is 10 ppm. This review aims at updating the available knowledge on the various detoxification cyanide in food.