A geminivirus-induced gene silencing system for gene function validation in cassava

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.     

Over-expression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification

Cassava (Manihot esculenta, Crantz) roots are the primary source of calories for more than 500 million people, the majority of whom live in the developing countries of Africa. Cassava leaves and roots contain potentially toxic levels of cyanogenic glycosides. Consumption of residual cyanogens (linamarin or acetone cyanohydrin) in incompletely processed cassava roots can cause cyanide poisoning. Hydroxynitrile lyase (HNL), which catalyses the conversion of acetone cyanohydrin to cyanide, is expressed predominantly in the cell walls and laticifers of leaves. In contrast, roots have very low levels of HNL expression. We have over-expressed HNL in transgenic cassava plants under the control of a double 35S CaMV promoter. We show that HNL activity increased more than twofold in leaves and 13-fold in roots of transgenic plants relative to wild-type plants. Elevated HNL levels were correlated with substantially reduced acetone cyanohydrin levels and increased cyanide volatilization in processed or homogenized roots. Unlike acyanogenic cassava, transgenic plants over-expressing HNL in roots retain the herbivore deterrence of cyanogens while providing a safer food product.     

Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers. Distribution of cyanogenic glucosides, their site of synthesis and transport, and blockage of the biosynthesis by RNA interference technology

Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (<1% of wild type) leaves were obtained by RNA interference to block expression of CYP79D1 and CYP79D2, the two paralogous genes encoding the first committed enzymes in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (<1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation.     

Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the cyanogenic glucosides lotaustralin and linamarin in Lotus japonicus

Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid l-Ile, whereas linamarin is derived from Val. In silico homology searches identified two cytochromes P450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with l-Ile as substrate than with l-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amounts of lotaustralin and rhodiocyanoside A/D were unaltered.     

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.     

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.     

Characterization and expression profile of two UDP-glucosyltransferases, UGT85K4 and UGT85K5, catalyzing the last step in cyanogenic glucoside biosynthesis in cassava

Manihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from l ‐valine and l ‐isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside‐specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2‐hydroxy‐2‐methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co‐occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co‐expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine‐tuning nitrogen assimilation in cassava.     

REVIEW ARTICLE: Cyanogenesis in cassava (Manihot esculenta Crantz)

Cassava is the most agronomically important of the cyanogeniccrops. Linamarin, the predominant cyanogenic glycoside in cassava,can accumulate to concentrations as high as 500 mg kg^–1fresh weight in roots and to higher levels in leaves. Recently,the pathway of linamarin synthesis and the cellular site oflinamarin storage have been determined. In addition, the cyanogenicenzymes, linamarase and hydroxynitrile lyase, have been characterizedand their genes cloned. These results, as well as studies onthe organ- and tissue-specific localization of linamarase andhydroxy-nitrile lyase, allow us to propose models for the regulationof cyanogenesis in cassava. There remain, however, many unansweredquestions regarding the tissue-specific synthesis, transport,and accumulation of cyanogenic glycosides. The resolution ofthe sequestions will facilitate the development of food processing,biochemical and transgenic plant approaches to reducing thecyanogen content of cassava foods. Key words: Cyanide, cyanogenic glycosides, linamarin, cyanogens     

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 β-glycosides in addition to linamarin. The enzyme is unusually stable and has a temperature optimum of 55°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.     

An enzyme-immobilized microplate for determination of linamarin for large number of samples

Summary An enzyme-immobilized microplate for determination of linamarin was prepared by covalently linking cassava leaf linamarase to the microplate. For linamarin determination, cassava roots were homogenised in 0.1 Mo-phosphoric acid and the filtrate adjusted to pH 6 with NaOH prior to adding into the wells. The cyanide released was then determined spectrophotometrically. One nmol linamarin can be detected. The microplate method is suitable for analysis of large number of samples and is useful for screening purposes.     

Transfer and Expression of an Artificial Storage Protein (ASP1) Gene in Cassava (Manihot Esculenta Crantz)

In order to increase the nutritional quality of cassava storage roots, which contain up to 85% starch of their dry weight, but are deficient in protein, a synthetic ASP1 gene encoding a storage protein rich in essential amino acids (80%) was introduced into embryogenic suspensions of cassava via Agrobacterium-mediated gene transfer. Transgenic plants were regenerated from suspension lines derived from hygromycin-resistant friable embryogenic callus lines. Molecular analysis showed the stable integration of asp1 in cassava genome and its expression at RNA level in transformed suspension lines. PCR and Southern analyses proved the transgenic nature of the regenerated plant lines. The expression of asp1 at RNA level was demonstrated by RT-PCR. The ASP1 tetramer could be detected in leaves as well as in primary roots of cultured transgenic plants by western blots. These results indicate that the nutritional improvement of cassava storage roots may be achieved by constitutive expression of asp1 in transgenic plants.     

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz)

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava’s starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

Metabolic engineering of indole glucosinolates in Chinese cabbage hairy roots expressing Arabidopsis CYP79B2, CYP79B3, and CYP83B1

Indole glucosinolates (IG), a group of secondary metabolites found almost extensively in the order Brassicales, play an important role in the interaction between plant and insect or microorganism. In order to explore the possibility of IG metabolic engineering in Chinese cabbage hairy roots, three Arabidopsis cDNAs CYP79B2, CYP79B3, and CYP83B1 combined with rolABC were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B2, CYP79B3, or CYP83B1 alone did not affect the accumulation levels of IG in transgenic hairy roots. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, some of the transgenic hairy roots accumulated higher levels of glucobrassicin (GBC) or 4-methoxy glucobrassicin (4-OMeGBC) than control hairy root line carrying rolABC vector. With regard to the IG accumulation, overexpression of all three cDNAs showed no better than overexpression of both cDNAs. Both 4-OMeGBC and neoglucobrassicin (neo-GBC) were found to be the main components of IG that comprise about 90% of total IG in all types of Chinese cabbage hairy root lines. In transgenic hairy root lines rB3B1-8 and rB2B1B3-5, 4-OMeGBC increased to 2 and 1.5-fold, while neo-GBC decreased to 0.5 and 0.6-fold, respectively. This suggests that an increased production of 4-OMeGBC causes a reduction of neo-GBC level since the two types derive from a common precursor GBC. However, in terms of the total IG level, the transgenic hairy roots did not show significant differences from controls.     

Cytotoxicity of purified cassava linamarin to a selected cancer cell lines

Cassava (Manihot esculenta Crantz) is a known source of linamarin, but difficulties associated with its isolation have prevented it from being exploited as a major source. A batch adsorption process using activated carbon proved successful in its isolation, with ultrafiltration playing a pivotal role in its purification. Thirty-two minutes of contact time was required for 60 g of extract, yielding 1.7 g of purified product. Picrate paper, infra-red and ¹HNMR analysis confirmed the presence and structure of linamarin. Cytotoxic effects of linamarin on MCF-7, HT-29 and HL-60 cells were determined using the MTT assay. Cytotoxic effects were significantly increased in the presence of linamarase (β-glucosidase), with a 10–fold decrease in the IC₅₀ values obtained for HL-60 cells. This study thus describes a method for the isolation and purification of linamarin from cassava, as well as its cytotoxicity potential.     

Development and application of transgenic technologies in cassava

The capacity to integrate transgenes into the tropical root crop cassava (Manihot esculenta Crantz) is now established and being utilized to generate plants expressing traits of agronomic interest. The tissue culture and gene transfer systems currently employed to produce these transgenic cassava have improved significantly over the past 5 years and are assessed and compared in this review. Programs are underway to develop cassava with enhanced resistance to viral diseases and insects pests, improved nutritional content, modified and increased starch metabolism and reduced cyanogenic content of processed roots. Each of these is described individually for the underlying biology the molecular strategies being employed and progress achieved towards the desired product. Important advances have occurred, with transgenic plants from several laboratories being prepared for field trails.