Cassava - The search for the genes encoding the biosynthetic enzymes
To understand the role of cyanogenic glucosides in cassava (Manihot esculenta
Crantz) the expression patterns of the genes involved in the biosynthesis are being examined during plant development and in response to abiotic and biotic stress factors like salinity, drought and viral attack. Next we want to characterise the promotors of the genes encoding the enzymes involved in the biosynthesis to investigate the regulation of the production of cyanogenic glucosides.
Cellular localisation and turnover of cyanogenic glucosides
We have cellularly localised the synthesis of the cyanogenic glucosides in the petiole and leaf blade of the first unfolded leaf by using in situ PCR and antibodies specific for the enzymes involved. In the petiole the synthesis is found in the cortex, endodermis and phloem and xylem parenchyma cells.
Localisation of the degrading enzymes linamarase and α-hydroxy nitrile lyase (α-HNL). Does this enzyme localisation determine if the cultivar is sweet or bitter?
A previous study showed a localisation of linamarase to the laticifer cells of cassava and a differential distribution of linamarase was found at the subcellular level between high and low cyanide containing cultivars (Santana et al., 2002). Transport of linamarase from shoot to root was suggested as no transcript of linamarase was detected in the roots of both cultivars. A similar difference of localisation of β-glucosidase activity between bitter and sweet varieties was also found in almonds (Sánchez-Pérez et al., 2008).
Our aim is to investigate the turnover of cyanogenic glucosides in different cassava cultivars and determine whether the difference in high- and low-cyanide cultivars is to some extent due to a difference in localisation and regulation of the breakdown enzymes, linamarase and α-HNL.
The aim of the project is to use molecular breeding to produce cassava plants devoid of or with a tissue-specific reduction in their cyanogenic glucoside content. Cassava is the most important root crop and the third most important staple in the Tropics. Especially under adverse conditions, cassava provides food security to the World’s poorest people.
The cyanogenic nature of cassava renders it necessary to carefully process roots and leaves before use. When the processing is not properly conducted, residual amounts of cyanide in cassava products may present a health hazard to the consumer. Proper processing results in a simultaneous partial loss of proteins, minerals and vitamins.
Breeding towards cassava varieties with an elevated content of proteins and vitamins has little benefit as long as subsequent processing results in loss of these valuable components and recovery of a starch product only. To overcome these problems, we want to block cyanogenic glucoside formation in cassava in the entire plant or in specific tissues.
Molecular breeding to improve the nutritional value of cassava to produce plants that do not liberate cyanide. Transgenic cassava is produced using RNA interference technology. The synthesis of cyanogenic glucosides is blocked either in the entire plant or in specific tissues. Additional nutritional traits will be introduced by gene pyramiding.
Cassava contains the two cyanogenic glucosides linamarin and lotaustralin derived from valine and isoleucine, respectively. We have isolated and cloned the two genes CYP79D1 and CYP79D2 encoding the enzymes that catalyse the first biosynthetic step.
Using a protocol based on Agrobacterium tumefaciens mediated transformation of the cassava cultivar Mcol22 via organogenesis, post transcriptional gene silencing (RNA interference) of the CYP79D1 and CYP79D2 genes has been accomplished. This resulted in a 98% reduction in cyanogenic glucoside content in young cassava plants compared to the wild type. These acyanogenic lines are being used as starting material for the introduction of other desired traits such as virus resistance by gene pyramiding.
Jørgensen K , Morant AV, Morant M , Jensen NB , Olsen CE , Kannangara RM et al . Biosynthesis of the cyanogenic glucosides linamarin and lotaustralin in cassava: Isolation, biochemical, characterization, and expression pattern of CYP71E7, the oxime-metabolizing cytochrome P450 enzyme . Plant Physiology . 2011;155:282-292.