Cassava Storage Roots Research Articles

Molecular cloning and characterization of a novel RING zinc-finger protein gene up-regulated under in vitro salt stress in cassava

Cassava (Manihot esculenta Crantz) is one of the world's most important food crops. It is cultivated mainly in developing countries of tropics, since its root is a major source of calories for low-income people due to its high productivity and resistance to many abiotic and biotic factors. A previous study has identified a partial cDNA sequence coding for a putative RING zinc finger in cassava storage root. The RING zinc finger protein is a specialized type of zinc finger protein found in many organisms. Here, we isolated the full-length cDNA sequence coding for M. esculenta RZF (MeRZF) protein by a combination of 5' and 3' RACE assays. BLAST analysis showed that its deduced amino acid sequence has a high level of similarity to plant proteins of RZF family. MeRZF protein contains a signature sequence motif for a RING zinc finger at its C-terminal region. In addition, this protein showed a histidine residue at the fifth coordination site, likely belonging to the RING-H2 subgroup, as confirmed by our phylogenetic analysis. There is also a transmembrane domain in its N-terminal region. Finally, semi-quantitative RT-PCR assays showed that MeRZF expression is increased in detached leaves treated with sodium chloride. Here, we report the first evidence of a RING zinc finger gene of cassava showing potential role in response to salt stress.

Iron Biofortification and Homeostasis in Transgenic Cassava Roots Expressing the Algal Iron Assimilatory Gene, FEA1.

We have engineered the tropical root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory gene, FEA1, in its storage roots with the objective of enhancing the root nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 g meal. Significantly, the expression of the FEA1 gene in storage roots did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of ferrous iron mediated by the FEA1 protein. Relative to wild-type plants, fibrous roots of FEA1 expressing plants had reduced Fe (III) chelate reductase activity consistent with the more efficient uptake of iron in the transgenic plants. We also show that multiple cassava genes involved in iron homeostasis have altered tissue-specific patterns of expression in leaves, stems, and roots of transgenic plants consistent with increased iron sink strength in transgenic roots. These results are discussed in terms of strategies for the iron biofortification of plants.

Increased storage protein from interspecific F1 hybrids between cassava (Manihot esculenta Crantz) and its wild progenitor (M. esculenta ssp. flabellifolia)

Cassava is an important food security crop in the developing world, as it is adapted to a wide range of climatic conditions including marginal semi-arid agro-ecologies. Cassava is a starchy staple and the storage roots of commercial cassava cultivar are very low in protein content (0.5–2%, dry weight basis). A diet predominantly based on cassava, as is the case in several sub-Saharan countries, could lead to malnutrition, especially in young children over time. A wild progenitor of cassava, Manihot esculenta ssp. flabellifolia have been found to have high root protein content, up to 18% (dry weight basis) and is excellent source of genetic variability for this important trait. Accessions of M. esculenta ssp. flabellifolia with high storage root protein content were crossed with commercial cassava cultivars. High content of storage root protein ranged from 2.87 to 11.25% could be recovered in the F1 progenies. The F1 families had an average dry matter content of 29.6%. These F1 interspecific hybrids would be an entry point for improvement of cassava storage root protein content. A brief discussion of the strategy to be followed is presented.

Expression Profiling of Cassava Storage Roots Reveals an Active Process of Glycolysis/GluconeogenesisF

Mechanisms related to the development of cassava storage roots and starch accumulation remain largely unknown. To evaluate genome-wide expression patterns during tuberization, a 60 mer oligonucleotide microarray representing 20 840 cassava genes was designed to identify differentially expressed transcripts in fibrous roots, developing storage roots and mature storage roots. Using a random variance model and the traditional twofold change method for statistical analysis, 912 and 3 386 upregulated and downregulated genes related to the three developmental phases were identified. Among 25 significantly changed pathways identified, glycolysis/gluconeogenesis was the most evident one. Rate-limiting enzymes were identified from each individual pathway, for example, enolase, L-lactate dehydrogenase and aldehyde dehydrogenase for glycolysis/gluconeogenesis, and ADP-glucose pyrophosphorylase, starch branching enzyme and glucan phosphorylase for sucrose and starch metabolism. This study revealed that dynamic changes in at least 16% of the total transcripts, including transcription factors, oxidoreductases/transferases/hydrolases, hormone-related genes, and effectors of homeostasis. The reliability of these differentially expressed genes was verified by quantitative real-time reverse transcription-polymerase chain reaction. These studies should facilitate our understanding of the storage root formation and cassava improvement.

Increased productivity through integrated soil fertility management in cassava–legume intercropping systems in the highlands of Sud-Kivu, DR Congo

Smallholder farmers in sub-Saharan Africa are confronted by low productivity and limited investment capacity in nutrient inputs. Integrated soil fertility management (ISFM) aims at increased productivity through the combined use of improved germplasm, judicious fertilizer application and organic matter management, adapted to the local farming conditions. We hypothesize that the application of these different ISFM components can result in significant increases in productivity and economic benefits of cassava–legume intercropping systems. Participatory demonstration trials were conducted in the highlands of Sud-Kivu, DR Congo with 12 farmer groups during 3 seasons. Treatments included the farmers’ common practice (local common bean and cassava varieties, seed broadcast and manure addition) and sequentially added ISFM components: improved bean and cassava germplasm, modified crop arrangements, compound NPK fertilizer application and alternative legume species (groundnut or soybean). The use of improved germplasm did not result in yield increases without simultaneous implementation of other ISFM components. Modifying the crop arrangement by planting cassava at 2 m between rows and 0.5 m within the row, intercropped with four legume lines, increased bean yields during the first season and permits a second bean intercrop, which can increase total legume production by up to 1 t ha −1 and result in an additional revenue of almost 1000 USD ha −1. Crop arrangement or a second legume intercrop did not affect cassava storage root yields. Fertilizer application increased both legume and cassava yield, and net revenue by 400–700 USD ha −1 with a marginal rate of return of 1.6–2.7. Replacing the common bean intercrop by groundnut increased net revenue by 200–400 USD ha −1 partly because of the higher market value of the grains, but mostly due to a positive effect on cassava storage root yield. Soybean affected cassava yields negatively because of its high biomass production and long maturity period; modifications are needed to integrate a soybean intercrop into the system. The findings demonstrate the large potential of ISFM to increase productivity in cassava–legume systems in the Central-African highlands. Benefits were, however, not observed in all study sites. In poor soils, productivity increases were variable or absent, and soil amendments are required. A better understanding of the conditions under which positive effects occur can enable better targeting and local adaptation of the technologies.

Characterisation and role of <i>Isoamylase1 (Meisa1</i>) gene in cassava

The current concept for starch biosynthesis in plants is that amylopectin, the major fraction of starch, is synthesised by the concerted actions of ADP-Glc pyrophosphorylase (AGPase), soluble starch synthase (SS), starch-branching enzyme (BE), and starch-debranching enzyme (DBE). We have isolated a cDNA clone ofIsoamylase1 gene, a member of DBE family from cassava (Manihot esculenta Crantz) storage root. The cloned cDNA fragment sequence (764 bp) showed high identity of 90% to Rcisa1, and 81% identity to Psisa1, Stisa1 and Atisa1. The deduced protein sequence showed highest (92%) identity with RCISA1. The comparative sequence analysis confirmed the cloned fragment to be M. esculenta isoamylase1 gene (Meisa1; accession number GU229751). Genomic analysis revealed occurrence of at least two copies of the Meisa1 gene. HighestMeisa1 transcript expression levels were detected in fibrous root followed by in the stem and least detected in the leaf and petiole. Analysis of the temporal expression pattern in the storage root showed initial and maximum expression at 90 days after planting (DAP), and declined thereafter to undetectable levels by 180 DAP. The results implicate a major role of Meisa1 in storage root differentiation and early starch granule initiation.

Changes in total carotenoid content at different stages of traditional processing of yellow‐fleshed cassava genotypes

AbstractThe changes in content of total carotenoid at each stage of processing cassava storage roots were investigated with three improved yellow‐fleshed cassava varieties (TMS 94/0006, TMS 01/1235 and TMS 01/1371) grown in 2005/2006 in a randomised complete block design with two replications at Ibadan, Nigeria. When the cassava roots were grated to a mash, results obtained indicate that there was a significant reduction in total carotenoid content for all the genotypes. The reduction was highest for TMS 01/1235 (1.20 μg g−1), intermediate for TMS 01/1371 (0.78 μg g−1) and least for TMS 94/0006 (0.35 μg g−1). In most cases, we observed higher total carotenoid concentration, especially when the intermediate step involved pressing to remove excess water, and during roasting compared with the initial concentration in the raw cassava storage roots. In conclusion, grating, drying and cooking to a paste resulted in reduction of total carotenoid content, while roasting and pressing resulted in higher carotenoid concentration. A change in total carotenoid content during processing depends on variety, processing method, especially unit operation and the initial total carotenoid content of the variety.

Quality evaluation of ‘lafun’ produced from stored cassava roots

Freshly harvested cassava roots were stored in the soil and some were stored heaped on the floor of a well ventilated store room. These roots were stored for periods ranging from 1 to 9 days and samples were drawn from them respectively at 3 days interval and used to produce Lafun. Also, Lafun was produced from freshly harvested cassavaroots. The proximate composition, physicochemical characteristics, rheological characteristics and sensory quality of the Lafun produced were evaluated. The moisture content of the cassava roots decreased with storage period, irrespective of the storage conditions, from 67.75% in the fresh cassava to 55.61% in the stored cassava roots. The proximate composition of Lafun obtained from freshly harvested cassava roots were higher than those obtainedfrom the stored roots and the observed differences were more marginal with soil stored cassava roots than room stored cassava roots. The quantity of lafun produced from the stored cassava roots decreased with storage. The percentage reduction in yield varied from 8.2% to 25% over the 9 days storage period in the room, and from 2.5% to 5.6% over the 9 days storageperiod in the soil. Lafun produced from cassava roots storedin the soil for 1 to 6 days and cassava roots stored in the room for 1 to 3 days had final viscosity (326.75 –333.25RVU), Water Absorption Capacity (81.91 –91.81ml/gm), Bulk Density (0.55 – 0.58mg/ml) andSwelling Index (3.87 – 3.91) which were respectively similar to those obtained from freshly harvested cassava roots. However, lower values were obtained respectively, from Lafun produced from 9 days soil stored and 6 days to 9 days room stored cassava roots. There were no significant differences (P

Isolation and characterization of the promoter sequence of a cassava gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in storage roots

Cassava is one of the most important tropical food crops for more than 600 million people worldwide. Transgenic technologies can be useful for increasing its nutritional value and its resistance to viral diseases and insect pests. However, tissue-specific promoters that guarantee correct expression of transgenes would be necessary. We used inverse polymerase chain reaction to isolate a promoter sequence of the Mec1 gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in cassava storage roots. In silico analysis revealed putative cis-acting regulatory elements within this promoter sequence, including root-specific elements that may be required for its expression in vascular tissues. Transient expression experiments showed that the Mec1 promoter is functional, since this sequence was able to drive GUS expression in bean embryonic axes. Results from our computational analysis can serve as a guide for functional experiments to identify regions with tissue-specific Mec1 promoter activity. The DNA sequence that we identified is a new promoter that could be a candidate for genetic engineering of cassava roots.

Influence of Cassava Storage Root Development and Environmental Conditions on Starch Granule Size Distribution

AbstractAmong starch producing crops, cassava has a higher carbohydrate production than others under suboptimal conditions, more than 163×106 t of cassava starch are produced world‐wide each year and are used for direct human consumption, animal feeds and as raw material for a wide range of industrial products. A basic knowledge of starch characteristics and storage process is required to improve cassava starch quality and quantity. This report describes a comparative study of morphology, starch granule size distribution and amylose content in cassava storage roots grown under different conditions. Microscopic evaluation reveals that storage roots contain larger starch granules than fibrous roots. The study on three Asian cassava cultivars demonstrates the increment of starch granule size from outermost to innermost layers of storage root, while no significant change is observed in amylose content. The effects of developmental stage and growing season on cassava starch reveals that granule size is significantly influenced by both factors, while the amylose contents only fluctuate in a narrow range. The size of the starch granules increases particularly in the first six months after cultivation. Planting cassava in the beginning of the wet season in Asia yields larger granule sizes than planting in the dry season. The significance of this study lies in better understanding optimal growing conditions for improved starch characteristics and production, leading to a better quality agricultural production of this important food and materials crop.

Dry matter production and distribution in three cassava (Manihot esculenta Crantz) cultivars during the second vegetative plant cycle

A study was carried out in Araruna County, State of Paraná, to understand the relationship between the total dry matter yield and its proportion allocated to the storage roots of cassava (Manihot esculenta Crantz) plants in the second vegetative cycle. The experimental design was a randomized complete block in split-plot scheme with four replications. The plots consisted of the Mico, IAC 13 and IAC 14 cultivars and the monthly harvesting dates were assessed in the sub-plots. The results showed that the Mico and IAC 13 cultivars were more efficient in allocating dry matter to the storage roots. The IAC 14 cultivar allocated a higher proportion of assimilates to stems compared with the other two cultivars. With regard to the influence of harvesting time, the lowest harvest indexes were observed in the periods of more intense vegetative growth. However, the highest carbohydrate proportions were allocated to the storage roots during periods of low vegetative growth.

Sugar-mediated semidian oscillation of gene expression in the cassava storage root regulates starch synthesis

Starch branching enzyme (SBE) activity in the cassava storage root exhibited a diurnal fluctuation, dictated by a transcriptional oscillation of the corresponding SBE genes. The peak of SBE activity coincided with the onset of sucrose accumulation in the storage, and we conclude that the oscillatory mechanism keeps the starch synthetic apparatus in the storage root sink in tune with the flux of sucrose from the photosynthetic source. When storage roots were uncoupled from the source, SBE expression could be effectively induced by exogenous sucrose. Turanose, a sucrose isomer that cannot be metabolized by plants, mimicked the effect of sucrose, demonstrating that downstream metabolism of sucrose was not necessary for signal transmission. Also glucose and glucose-1-P induced SBE expression. Interestingly, induction by sucrose, turanose and glucose but not glucose-1-P sustained an overt semidian (12-h) oscillation in SBE expression and was sensitive to the hexokinase (HXK) inhibitor glucosamine. These results suggest a pivotal regulatory role for HXK during starch synthesis. Abscisic acid (ABA) was another potent inducer of SBE expression. Induction by ABA was similar to that of glucose-1-P in that it bypassed the semidian oscillator. Both the sugar and ABA signaling cascades were disrupted by okadaic acid, a protein phosphatase inhibitor. Based on these findings, we propose a model for sugar signaling in regulation of starch synthesis in the cassava storage root.

Feeding activity of the East African millipede Omopyge sudanica Kraus on different crop products in laboratory experiments

Millipedes can cause considerable damage in the production of sweet potato and some other crops in East Africa. Quantitative information on intake of crop diets by and body weight gain of millipedes was collected in short-term no-choice feeding activity laboratory experiments conducted in north-eastern Uganda using female millipedes of the species Omopyge sudanica. Diets consisted of sweet potato and cassava storage root material, groundnut seeds, or maize grains. Differences in intake and body weight gain between diets were not statistically different. The consumption index, i.e., the ratio between intake and body weight gain, was significantly higher for sweet potato than for most other diets. The efficiency of conversion of ingested food, i.e., 100 × the ratio between body weight gain and intake, was significantly lower for the root crops — especially sweet potato — than for the grain crops. The research showed how difficult it is to obtain reliable, quantitative data on the feeding habits of millipedes, but also illustrated that O. sudanica can cause harm to crops in north-eastern Uganda and elsewhere in East Africa.

Towards identifying the full set of genes expressed during cassava post-harvest physiological deterioration

Storage roots of cassava (Manihot esculenta Crantz) exhibit a rapid post-harvest physiological deterioration (PPD) response that can occur within 24-72 h of harvest. PPD is an enzymatically mediated oxidative process with parallels to plant wound, senescence and defence responses. To characterise those genes that show significant change in expression during the PPD response we have used cDNA microarray technology to carry out a large-scale analysis of the cassava root transcriptome during the post-harvest period. We identified 72 non-redundant expressed sequence tags which showed altered regulation during the post-harvest period. Of these 63 were induced, whilst 9 were down-regulated. RNA blot analysis of selected genes was used to verify and extend the microarray data. Additional microarray hybridisation experiments allowed the identification of 21 root-specific and 24 root-wounding-specific sequences. Many of the up-regulated and PPD-specific expressed sequence tags were predicted to play a role in cellular processes including reactive oxygen species turnover, cell wall repair, programmed cell death, ion, water or metabolite transport, signal transduction or perception, stress response, metabolism and biosynthesis, and activation of protein synthesis.

A cDNA Sequence Coding for a Glutamic Acid-Rich Protein Is Differentially Expressed in Cassava Storage Roots

We report the isolation and characterization of a cDNA sequence (Mec1) coding for a glutamic acid-rich protein (Pt2L4) from cassava storage roots. Comparative sequence analysis showed a high identity of Pt2L4 with cassava protein C54, which is expressed in vascular tissues of storage roots. Northern blot analysis showed that the Mec1 transcript expression pattern might be related to the maturation of the storage parenchyma cells.

Comparative Gene Expression Study to Identify Genes Possibly Related to Storage Root Formation in Cassava

Cassava storage roots result from swelling of adventitious roots by secondary growth. In the present study we aimed to gain insight into the molecular processes occurring during cassava storage root formation. We report a comparative gene expression study in adventitious and storage roots in order to identify genes possibly related to storage organ formation. Our results revealed five genes with higher expression levels in secondary xylem of storage roots than adventitious roots. Among them, the Mec1 gene coding for Pt2L4 glutamic acid-rich protein and a putative RING Zinc Finger and LEA protein genes were strongly induced in secondary xylem tissue.

Studies toward the identification of transcription factors in cassava storage root

Transcription factors play important roles in several physiological processes. In recent years many transcription factors have been isolated from plants and they are emerging as powerful tools in the manipulation of plant traits. In this work we initiated studies in order to isolate transcription factors from cassava (Manihot esculenta Crantz), an important tropical and subtropical crop. Our results show three kinds of proteins expressed differentially in cassava storage root and immunologically related to the opaque-2 transcription factor from maize. Southwestern experiments showed two proteins capable of interacting in vitro with the DNA sequence of the be2S1 gene from the Brazil nut tree.

Two cassava promoters related to vascular expression and storage root formation.

Cassava ( Manihot esculenta Crantz) storage roots, organs accumulating large amounts of starch, develop from primary roots via secondary growth. The availability of promoters related to storage-root formation is a prerequisite for engineering root traits in cassava. Two cDNAs, c15 and c54, were identified from a storage-root cDNA library of cassava MCol1505 via differential screening. The transcripts of c15 and c54 were detected in storage roots but not in leaves by Northern analysis. Homology analysis of the deduced amino acid sequences showed that C15 is likely to be related to cytochrome P450 proteins, which are involved in the oxidative degradation of various compounds, while C54 may be related to Pt2L4, a cassava glutamic acid-rich protein. The promoter regions of c15 and c54 were isolated from the corresponding clones in a cassava genomic library. A 1,465-bp promoter fragment ( p15/1.5) of c15 and a 1,081-bp promoter region ( p54/1.0) of c54 were translationally fused to the uidA reporter gene, and introduced into cassava and Arabidopsis thaliana (L.) Heynh. The expression patterns of p15/1.5::uidA and p54/1.0::uidA in transgenic plants showed that both promoters are predominantly active in phloem, cambium and xylem vessels of vascular tissues from leaves, stems, and root systems. More importantly, strong beta-glucuronidase activity was also detected in the starch-rich parenchyma cells of transgenic storage roots. Our results demonstrate that the two promoters are related to vascular expression and secondary growth of storage roots in cassava.

Expression patterns of the gene encoding starch branching enzyme II in the storage roots of cassava ( Manihot esculenta Crantz)

Spatial and temporal expression patterns of the sbeII and sbeI genes, encoding starch branching enzyme II and I, respectively, in cassava ( Manihot esculenta Crantz) were studied at different phenological stages of the crop. A partial cDNA for sbeII in cassava was cloned and used along with a cDNA-specific fragment of sbeI. As the cassava plant aged, the transcriptional activity of the sbeII and sbeI genes in the underground storage roots increased, whereas the activity in other organs remained the same or declined. At 180 days after planting (d.a.p.), levels of sbeII and sbeI transcripts in storage roots were very low, whereas at 360 d.a.p., the levels had increased dramatically. The 360 d.a.p. old storage roots also accumulated gbssII and gbssI transcripts, as well as a longer gbssI transcript, gbssI′. The difference between the gbssI and gbssI′ transcripts was shown to be due to differential splicing, whereby the gbssI′ transcript retained the first three introns. Unexpectedly, expression of sbeII and sbeI in the 360 d.a.p. storage roots exhibited fluctuations during the 24 h cycle, both under the normal light/dark regime and under continuous light or continuous dark conditions.

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.