Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanide-induced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration.     

iTRAQ-based analysis of changes in the cassava root proteome reveals pathways associated with post-harvest physiological deterioration

The short storage life of harvested cassava roots is an important constraint that limits the full potential of cassava as a commercial food crop in developing countries. We investigated the molecular changes during physiological deterioration of cassava root after harvesting using isobaric tags for relative and absolute quantification (iTRAQ) of proteins in soluble and non‐soluble fractions prepared during a 96 h post‐harvest time course. Combining bioinformatic approaches to reduce information redundancy for unsequenced or partially sequenced plant species, we established a comprehensive proteome map of the cassava root and identified quantitatively regulated proteins. Up‐regulation of several key proteins confirmed that physiological deterioration of cassava root after harvesting is an active process, with 67 and 170 proteins, respectively, being up‐regulated early and later after harvesting. This included regulated proteins that had not previously been associated with physiological deterioration after harvesting, such as linamarase, glutamic acid‐rich protein, hydroxycinnamoyl transferase, glycine‐rich RNA binding protein, β‐1,3‐glucanase, pectin methylesterase, maturase K, dehydroascorbate reductase, allene oxide cyclase, and proteins involved in signal pathways. To confirm the regulation of these proteins, activity assays were performed for selected enzymes. Together, our results show that physiological deterioration after harvesting is a highly regulated complex process involving proteins that are potential candidates for biotechnology approaches to reduce such deterioration.     

Lysozyme inhibits postharvest physiological deterioration of cassava

After harvest, cassava (Manihot esculenta Crantz) storage roots undergo rapid postharvest physiological deterioration, producing blue-brown discoloration in the vasculature due to the production of polyphenolics (mainly quinones and coumarins) by enzymes such as polyphenol oxidase (PPO). Here, we report the application of hen egg-white lysozyme (HEWL), a natural PPO inhibitor, in transgenic cassava to repress the symptoms of postharvest physiological deterioration. The HEWL-expressing transgenic plants had lower levels of the two main cassava coumarins tested, scopoletin and scopolin, compared with wild type. HEWL-expressing cassava also showed increased tolerance of oxidative stress. Overall, the lysozyme-PPO system proved to be functional in plants for repressing PPO-mediated commercial product browning.     

Cassava postharvest physiological deterioration: a complex phenomenon involving calcium signaling,reactive oxygen species and programmed cell death

Postharvest physiological deterioration (PPD) of cassava (Manihot esculenta) storage roots is a complex physiological and biochemical process which involve many regulatory networks linked with specific proteins modulation and signaling transduction pathways. However, it is poorly understood regarding biological regulation, and the interactions among protein groups and signals to determine PPD syndrome in cassava storage roots. This review sheds some light on the possible molecular mechanisms involved in reactive oxygen species (ROS), calcium signaling transduction, and programmed cell death (PCD) in cassava PPD syndrome. A model for predicting crosstalk among calcium signaling, ROS and PCD is suggested to fine-tune PPD syndrome. This would clues to cassava molecular breeding to alleviate the PPD effects on the shelf-life.     

Comparative Transcriptomic Analysis of Storage Roots in Cassava During Postharvest Physiological Deterioration

Plant Molecular Biology Reporter - Cassava is an important starchy and food crop; however, the commercial value of cassava is seriously constrained by postharvest physiological deterioration (PPD)....     

Constituents and secondary metabolite natural products in fresh and deteriorated cassava roots

A phytochemical analysis of cassava (Manihot esculenta Crantz) fresh roots and roots suffering from post-harvest physiological deterioration (PPD) has been carried out. The first isolation and identification of galactosyl diacylglycerides from fresh cassava roots is reported, as well as β-carotene, linamarin, and β-sitosterol glucopyranoside. The hydroxycoumarin scopoletin and its glucoside scopolin were identified from cassava roots during PPD, as well as trace quantities of esculetin and its glucoside esculin. There is no isoscopoletin in cassava roots during PPD.     

木薯储藏根采后生理性变质研究进展

木薯(Manihot esculenta Crantz)是热带、亚热带地区重要的粮食作物和能源作物.木薯产量很高,储藏根富含淀粉,但收获后采后生理性变质严重,严重影响了木薯的开发和利用.结合近期研究工作,综述了木薯储藏根采后生理性变质的研究进展,包括采后生理性变质的检测标准、生化基础、抗采后生理性变质的杂交育种、以活性氧自由基为主要研究对象的功能基因组学与基因工程、应用前景及存在的问题,以期为木薯储藏根采后生理性变质的遗传改良提供参考.     

Cassava: an appraisal of its phytochemistry and its biotechnological prospects

The present state of knowledge of the phytochemistry of small molecules isolated from the roots and leaves of cassava, Manihot esculenta Crantz (Euphorbiaceae), is reviewed. Cassava roots are an important source of dietary and industrial carbohydrates, mainly eaten as a source of starch, forming the staple food to over 500 million; additionally, the roots have value as a raw material for industrial starch production and for animal feed giving the crop high economic value, but it suffers markedly from post-harvest physiological deterioration (PPD). The hydroxycoumarins scopoletin and its glucoside scopolin as well as trace quantities of esculetin and its glucoside esculin are identified from cassava roots during PPD. The biotechnological prospects for cassava are also reviewed including a critical appraisal of transgenic approaches for crop improvement, together with its use for bioethanol production, due to cassava's efficient ability to fix carbon dioxide into carbohydrate.     

木薯分子育种中若干基本科学问题的思考与研究进展

木薯作为全球重要的薯类作物,既是热带地区粮食安全的保障,也是重要的淀粉工业原材料,保障其稳产、高产、优质一直是育种家不变的研究主题．当前,木薯品种选育正处在从杂交育种转向分子育种的发展阶段,深入解析木薯特有的经济性状和生物学特点是利用生物技术进行遗传改良的基础．不同于谷物类作物,木薯光合同化物的转运和库源分配的调控机制必有其独特之处;同时,储藏根的“库容”直接影响其产量．作为热带作物,了解木薯对低温和干旱的响应可为改良其抗逆境能力提供理论依据．不同于其他薯类作物,木薯储藏根特有的“采后生理性变质”问题亟待解决,其发生和调控机制的解析对延长木薯货架期意义重大．随着分子生物学的发展,针对上述各方面的研究日益深入,不仅激发了感兴趣的公众对这些问题的认知和思考,也激励了科研人员不断努力寻找解析相关机制的方法,为最终通过分子育种手段改良木薯提供思路和技术方案,揭开木薯的层层“面纱”,推动木薯分子育种的发展．     

Changes of cyanide content and linamarase activity in wounded cassava roots

When cassava (Manihot esculenta Crantz) root was cut into blocks and incubated under laboratory conditions, the blocks showed more widespread and more even symptoms of physiological deterioration than those under natural conditions. Thus, the tissue block system has potential for biochemical studies of natural deterioration of cassava root. The changes in cyanide content and linamarase (linamarin β-d-glucoside glucohydrolase; EC 3.2.1.21) activity in various tissues during physiological deterioration were investigated. Total cyanide content increased in all parts of block tissue after 3-day incubation. The degree of increase in cyanide was most pronounced in white parenchymal tissue, 2 to 3 millimeters thick, next to the cortex (A-part tissue), where no physiological symptoms appeared. On the other hand, linamarase activity was decreased in all parts of block tissue after a 3-day incubation. A time course analysis of A-part tissue indicated a clear reciprocal relationship between changes in total cyanide and linamarase activity; total cyanide increased, while linamarase activity decreased. Free cyanide constituted a very small portion of the total cyanide and did not change markedly.     

Root-surface mycoflora of cassava (Manihot esculenta) and post harvest rot of the tubers

The root-surface mycoflora of cassava were isolated from roots washed in serial changes of sterile distilled water and plated out on potato-dextrose agar. A small group of fungi which included Aspergillus niger, Botryodiplodia theobromae, Fusarium solani, Penicillium javanicum, Penicillium sp., and Trichoderma sp. were found to be consistently associated with the root surface. While the isolates, B. theobromae and F. solani were found to be aggressively pathogenic on freshly harvested cassava tubers causing extensive rot, A. niger was only mildly so. The root-surface mycoflora, therefore, includes fungi which have been reported as the most important in postharvest deterioration of the tubers. The removal of the rhizoplane microflora by surface-sterilization using calcium hypochlorite or Clorox and subsequent incubation in loosely tied polyethylene bags extended the storage life of the tubers considerably.     

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.     

Oxidative stress responses during cassava post-harvest physiological deterioration

A major constraint to the development of cassava (Manihot esculenta Crantz) as a crop to both farmers and processors is its starchy storage roots' rapid post-harvest deterioration, which can render it unpalatable and unmarketable within 24–72 h. An oxidative burst occurs within 15 min of the root being injured, that is followed by the altered regulation of genes, notably for catalase and peroxidase, related to the modulation of reactive oxygen species, and the accumulation of secondary metabolites, some of which show antioxidant properties. The interactions between these enzymes and compounds, in particular peroxidase and the coumarin, scopoletin, are largely confined to the vascular tissues where the visible symptoms of deterioration are observed. These, together with other data, are used to develop a tentative model of some of the principal events involved in the deterioration process. Abbreviations: ACMV, African cassava mosaic virus; AFLP, amplified fragment length polymorphism; CAT, catalase; cDNA, complementary deoxyribonucleic acid; CIAT, International Centre for Tropical Agriculture; Cu/ZnSOD, copper/zinc superoxide dismutase; DAB, 3,3-diaminobenzidine tetrahydrochloride; DPPH, 1,1-diphenyl-2-picrylhydrazyl; FeSOD, iron superoxide dismutase; FW, fresh weight; GUS, -glucuronidase; HPTLC, high-performance thin-layer chromatography; HR, hypersensitive response; IEF-PAGE, isoelectric focusing polyacrylamide gel electrophoresis; MAS, marker-assisted selection; MeJa, methyl jasmonate; MnSOD, manganese superoxide dismutase; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); NBT, nitroblue tetrazolium; PAL, phenylalanine ammonia-lyase; PCD, programmed cell death; PCR, polymerase chain reaction; POX, peroxidase; PPD, post-harvest physiological deterioration; QTL, quantitative trait loci; ROS, reactive oxygen species; RT, room temperature; SAR, systemic acquired resistance; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase     

Early events in the life of apple roots: variation in root growth rate is linked to mycorrhizal and nonmycorrhizal fungal colonization

Early events of mycorrhizal and nonmycorrhizal fungal colonization in newly-emerging roots of mature apple (Malus domestica Borkh) trees were characterized to determine the relationship of these events to fine root growth rate and development. New roots were traced on root windows to measure growth and then collected and stained to quantify microscopically the presence of mycorrhizal and nonmycorrhizal fungal structures. Most new roots were colonized by either mycorrhizal or nonmycorrhizal fungi but none less 25 days old were ever internally colonized by both. Compared to nonmycorrhizal colonization, mycorrhizal colonization was associated with faster growing roots and roots that grew for a longer duration, leading to longer roots. While either type of fungi was observed in roots as soon as 3 days after root emergence, intraradical colonization by mycorrhizal fungi was generally faster (peaking at 7 to 15 days) than that by nonmycorrhizal fungi and often occurred more frequently in younger roots. Only 15 to 35% of the roots had no fungal colonization by 30 days after emergence. This study provides the first detailed examination of the early daily events of mycorrhizal and nonmycorrhizal fungal colonization in newly emerging roots under field conditions. We observed marked discrimination of roots between mycorrhizal and nonmycorrhizal fungi and provide evidence that mycorrhizal fungi may select for faster growing roots and possibly influence the duration of root growth by non-nutritional means.     

Biochemical Changes in Secondary Metabolites in Wounded and Deteriorated Cassava Roots

Bluish fluorescent and phenolic components were produced in cassava roots in response to cut- injury, and in relation to physiological deterioration and microbial deterioration. The former roved to consist of five coumarin components, the main three of which were scopoletin, scopolin and esculin, and the other two were scopoletin- and esculetin-containing conjugates. A main component of the latter was (+)-catechin. Some enzymes pertaining to the production of the secondary metabolites such as acid invertase, phenylalanine ammonia lyase and peroxidase were formed in cut-injured tissue and in non-infected tissue adjacent to the soft-rotten part.     

Performances of improved cassava (Manihot esculenta Crantz) cultivars against root rot disease and yield in cassava-maize intercropping systems under natural infection

This study examined the performances of 21 cassava cultivars in two cropping seasons on the field against root rot disease and the yield in cassava-maize intercrop. Data were collected on number of root/plant, weight of root (t/ha) and disease severity (DS) on rotted roots at 12 and 16 months after planting (MAP), respectively. There were significant (P ≤ 0.05) differences for DS at 12 and 16 MAP in both seasons with cultivar TMS 97/JW2 having the least DS score. TMS 97/JW2 was resistant to the root rot pathogen, while eleven other cultivars were moderately resistant to the disease at 16 MAP. There was no consistency in the roots weight for the cultivars over the two cropping seasons but higher roots weight was recorded at 16 MAP than 12 MAP with different cultivars having highest roots weight at these periods. Intercropping maize with cassava does not have any management potential on root rot development.