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.     

Interaction Between Root and Leaf Disease Development in Barley Cultivars after Inoculation with Different Isolates of Bipolaris sorokiniana

The relationship between root and leaf infection in 11 cultivars of barley ( Hordeum vulgare ) by different isolates of Bipolaris sorokiniana was investigated in young plants. Roots of 10-day-old seedlings, grown in filterpaper rolls, and the third leaf of 17-day-old seedlings were inoculated with the different isolates and a Disease Development Index (DDI) was calculated.
The rate of lesion development in leaves was higher than in roots, indicated by generally higher DDI after leaf inoculation than after root inoculation. Significant differences in resistance were found among the barley cultivars. Inoculation with different isolates of B. sorokiniana caused significant differences in DDI for both roots and leaves. In the leaves, but not in the roots, a significant cultivar–isolate interaction was found. No significant correlations, neither in isolate aggressiveness nor in cultivar reaction between root and leaf, were observed.     

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.     

In vitro standardisation of resistance screening methods in cassava against tuber rot disease

Cassava tuber rot caused by Phytophthora palmivora in growing regions of Tamil Nadu and Kerala, is causing yield loss up to 80%. In the present study, resistance reactions of 10 cassava cultivars were analysed on leaf, stem and tuberous roots by artificial inoculation method in search of a suitable in vitro resistant screening method. Leaf and tuber analysis showed positive correlation (0.883) but the stem-based results showed negative correlation with leaf and tuber analysis. The analysis exhibited the susceptibility of the cassava cultivars against P. palmivora. Leaf analysis was superior in discriminating even small variations in resistance reactions than tuber analysis. The cultivar Sree Padmanabha showed higher resistance than other cultivars and the level of resistance in a cultivar is heritable which could be helpful in breeding programme. Based on the results it can be concluded that leaves of cassava could be used for screening resistance in the host and also in analysing the virulence of the isolate. This is the first report on screening the resistance in cassava cultivars against root rot caused by P. palmivora.     

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.     

Water stress-induced alterations in the proline metabolism of drought-susceptible and -tolerant cassava (Manihot esculenta) cultivars

The free proline levels and activities of ornithine aminotransferase (EC 2.6.1.13) and proline oxidase (EC 1.5.2.2), two of the enzymes involved in proline metabolism were studied during the induction of water stress in a drought susceptible (M-4) and a drought tolerant (S-1315) cultivar of cassava ( Manihot esculenta Crantz). Water stress induced by polyethylene glycol (MW 6000, osmotic potential — 1.65 MPa) caused a ca 25-fold increase in proline in young excised leaves of the susceptible cultivar (M-4) while the increase was about 9-fold in the tolerant cultivar (S-1315). The activity of ornithine aminotransferase (OAT), a key enzyme involved in the biosynthesis of proline, was found to increase 3-fold in water stressed leaves of M-4 and about 2-fold in those of S-1315. The activity of proline oxidase, which is involved in the degradation of proline to pyrroline-5-carboxylate, was reduced by 50% in M-4 and nearly 25% in S-1315 on water stress. Comparison of the kinetic properties of OAT showed that the enzyme from water-stressed leaves is more stable to heat inactivation compared to that of control. These results indicate that during water stress there are alterations in the metabolism of proline in cassava, and the extent of alteration varies between drought-susceptible and -tolerant cultivars.     

Cyanogenic potential in cassava and its influence on a generalist insect herbivore Cyrtomenus bergi (Hemiptera: Cydnidae)

The hypothesis that cyanogenic potential in cassava is a defense mechanism against arthropod pests is one of the crucial questions relevant to current efforts to reduce or eliminate cyanogenic potential (CNP) in cassava. The generalist arthropod Cyrtomenus bergi, which attacks cassava roots, was used in a bioassay relating oviposition and survival to CNP, concentration of nonglycosidic cyanogens, and linamarase (beta-glycosidase) activity in twelve selfed cassava siblings and their parental clone, which has segregated for different levels of cyanogenesis. Electron microscopic evaluation revealed an intracellular pathway of the stylet of C. bergi in the cassava root tissue to rupture cell walls. This feeding behavior causes cyanogenesis and increased linamarin content in the hemolymph of C. bergi while feeding on a cyanogenic diet. This diet resulted in a significant reduction in oviposition, especially at levels of CNP above 150 ppm (expressed as hydrogen cyanide) on fresh weight basis (or 400 ppm on dry weight basis) in cassava roots. An exponential decline in oviposition was observed with increasing levels of CNP, beginning 12 d after exposure to the cyanogenic diet. Cyanogenic potential and dry matter content showed a positive effect on survival. No relationship was found between concentrations of nonglycosidic cyanogens or linamarase activity in the cassava root and either oviposition or survival. According to our results, there is a significant difference between potentially noncyanogen and high cyanogen clones, but there may not be a significant difference between potentially noncyanogen and low cyanogen clones. Consequently, more frequent outbreaks or higher levels of damage might not be anticipated in potentially noncyanogen cassava clones than that anticipated in low cyanogenic clones. The negative effect of cyanogenesis on oviposition concurrent with a positive effect on survival of this pest is most likely the result of a physiological trade-off between survival and oviposition. The question of whether ovipositional rates could be recovered after a long-term exposure to cyanide remains unanswered.     

In-situ localization of cyanogenic β-glucosidase (linamarase) gene expression in leaves of cassava (Manihot esculenta Cranz) using non-isotopic riboprobes

The release of hydrogen cyanide (cyanogenesis) from damaged plant tissue depends upon the sequential action of a β-glucosidase and an α-hydroxynitrilase on cyanoglucosides. The non-isotopic digoxigenin labelling system was used to visualize the presence of cyanogenic β-glucosidase (linamarase) mRNA in cells of young leaves of Manihot esculenta Cranz (cassava). Strong hybridization to antisense riboprobes produced from the cDNA clone pCAS5, indicates localization of linamarase gene expression in laticifers (latex vessels). This is supported by the demonstration of linamarase mRNA in exuded latex. In contrast, in-situ localization of the control gene pGLF4, showed expression in all leaf mesophyll cells. High levels of linamarase activity were demonstrated in the latex of leaf petioles and this activity was shown to be dependent on the presence of attached leaflets. Assays of α-hydroxynitrilase activity in exuded latex and whole leaves shows that, unlike linamarase, this enzyme is present at very low levels in latex and must be located elsewhere in the leaf.     

Activities of Glycolytic Enzymes in Leaves and Roots of Contrasting Cultivars of Sorghum During Flooding

Activities of phosphofructokinase (PFK), fructose-1,6-bisphosphate aldolase (FBP aldolase) and pyruvate kinase (PK) increased progressively in the roots of flood-tolerant SSG-59-3 cultivar during flooding. In contrast, only a slight change in activities of PFK and FBP aldolase was discerned in the roots of flood-sensitive S-308 cultivar during initial stages of flooding followed by a decline in the activities of these enzymes. Although the activity of hexokinase (HK) was transiently elevated in roots of both the cultivars during flooding, the magnitude of increase was much more in SSG-59-3 than in the S-308. In leaves of SSG-59-3, HK activity increased during 12 h of flooding whereas only a minor change occurred in the case of S-308. Flooding resulted in depressed activities of PFK and PK in leaves of S-308 but that in SSG-59-3 rose following imposition of waterlogged conditions. Activity of FBP aldolase in leaves of tolerant cultivar also showed a steady enhancement during flooding. The total and reducing sugars content decreased in leaves and roots of the S-308 during flooding but in SSG-59-3 the amount was more or less comparable to that in corresponding non-flooded plants.     

Remediation of Nickel-Contaminated Soil by Brassica juncea L. cv. T-59 and Effect of the Metal on Some Metabolic Aspects of the Plant

Among four cultivars of Brassica juncea L., viz., TM-4, TM-2, RH-30, and T-59, cv. T-59 was relatively more tolerant to nickel (Ni) toxicity based on the growth parameters, seedling vigor index, and metal tolerance index. Nickel application inhibited the activity of the nitrate-assimilating enzyme nitrate reductase in the roots, stem, and leaves, whereas the total organic nitrogen, proline, and activity of a polyamine-metabolizing enzyme, diamine oxidase, increased in this tolerant cultivar (T-59). It accumulated a good amount of Ni from the soil in its root and shoot (i.e., 6.0–6.51 μg Ni g^?1 dry weight) during 2 months of cultivation with an 8.0 mM Ni supply in the soil. The data presented in this paper indicate that Ni tolerance and its removal by Indian mustard from subtropical Indian soil is cultivar dependent, possibly due to different genetic and physiological adaptations of the cultivars.     

干旱胁迫对苗期大豆异黄酮合成的影响

以不同耐旱性的2个大豆品种(高耐旱JP-6、低耐旱JP-16)为研究材料,采用高效液相色谱和实时荧光定量PCR技术,分析不同时间持续干旱胁迫下,大豆叶片和根系中异黄酮的积累变化及关键酶基因的表达情况.结果表明:大豆根部异黄酮含量显著高于叶部,而异黄酮关键酶基因的表达量则在叶片中更高,耐旱品种JP-6根部的异黄酮积累量更大.随着干旱胁迫持续时间的增加,不同耐旱品种的异黄酮合成与积累变化规律存在显著差异:强耐旱品种JP-6的根和叶中,异黄酮积累量均呈现先下降后升高的趋势;而弱耐旱品种JP-16则相反,异黄酮积累量在不同部位中均呈现先上升后降低的趋势;除JP-6叶中C4H、4CL、IFS2等异黄酮合成上游基因外,其他不同品种、不同部位的关键酶基因表达量均随着干旱胁迫持续时间的增加,呈现先下降后上升的趋势.大豆叶片是异黄酮的主要合成部位,大豆根部也存在少量的异黄酮合成.弱耐旱大豆根部的异黄酮合成和最终积累量均较低,强耐旱品种则较高.根部异黄酮积累量高的大豆品种,其耐旱性更强.     

Silicification in sorghum (Sorghum bicolor) cultivars with different drought tolerance

Sorghum belongs to a group of economically important, silicon accumulating plants. X-ray microanalysis coupled with environmental scanning electron microscopy (ESEM) of fresh root endodermal and leaf epidermal samples confirms histological and cultivar specificity of silicification. In sorghum roots, silicon is accumulated mostly in endodermal cells. Specialized silica aggregates are formed predominantly in a single row in the form of wall outgrowths on the inner tangential endodermal walls. The density of silica aggregates per square mm of inner tangential endodermal cell wall is around 2700 and there is no significant difference in the cultivars with different content of silicon in roots. In the leaf epidermis, silicon deposits were present in the outer walls of all cells, with the highest concentration in specialized idioblasts termed 'silica cells'. These cells are dumb-bell shaped in sorghum. In both the root endodermis and leaf epidermis, silicification was higher in a drought tolerant cultivar Gadambalia compared with drought sensitive cultivar Tabat. Silicon content per dry mass was higher in leaves than in roots in both cultivars. The values for cv. Gadambalia in roots and leaves are 3.5 and 4.1% Si, respectively, and for cv. Tabat 2.2 and 3.3%. However, based on X-ray microanalysis the amount of Si deposited in endodermal cell walls in drought tolerant cultivar (unlike the drought susceptible cultivar) is higher than that deposited in the leaf epidermis. The high root endodermal silicification might be related to a higher drought resistance.     

Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz)

In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.     

干旱胁迫下小麦幼苗根、叶多胺含量和多胺氧化酶活性的变化

 以抗旱性不同的两个小麦品种（‘晋麦33’和‘温麦8’）（Triticum aestivum cv. Jinmai 33 and Wenmai 8）为材料,研究了干旱胁迫下多胺含量和多胺氧化酶活性的变化。结果表明：旱过程中,幼苗根、叶中腐胺（Put）、亚精胺（Spd）、精胺（Spm）3种多胺含量和多胺氧化酶（PAO）活性先迅速升高,而后下降。与抗旱性弱的‘晋麦33’相比,抗旱性强的品种‘温麦8’幼苗根、叶中Spd、Spm 含量初期升高幅度大,之后下降速率减慢;PAO活性的变化与之相反,‘晋麦33’的PAO活性提高的幅度大于‘温麦8号’。多胺含量和PAO活性在小麦幼苗的根与叶之间呈极显著正相关。干旱初期,小麦根、叶中多胺迅速积累可能是干旱胁迫反应的一个信号,随后较高的Spd、Spm 水平有利于增强小麦幼苗的抗旱性。     

Metabolic profiles of six African cultivars of cassava (Manihot esculenta Crantz) highlight bottlenecks of root yield

Cassava is an important staple crop in sub‐Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C₃ mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin–Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse‐grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.     

松嫩平原不同株型玉米品种根系分布特征比较研究

采用土柱模拟栽培法与大田试验相结合的方法,对松嫩平原不同株型玉米的根系分布特征进行了比较。结果表明,平展型玉米和紧凑型玉米根干重最大值出现的时期不同,二者根干重分别在抽丝后15d和抽丝后30d时达到最大值,成熟时紧凑型玉米根干重比平展型高12．2％,二者根系垂直分布有明显的差异,在20cm以下的根干重比率,平展型玉米在19％以下,而紧凑型玉米高于23％,紧凑型玉米的深层根量较多,在深40～100cm土层内根干重比率比平展型高42．3％,二者的根系水平分布也不同,紧凑型玉米根系水平分布较集中,在距植株0～10cm水平范围内,根系分布比率比平展型玉米高9．6％,紧凑型玉米深层根量较多,水平分布集中,耐密植,是易获得高产的重要原因之一。     

Potato Responds to Salt Stress by Increased Activity of Antioxidant Enzymes

To understand the response of potato to salt stress, antioxidant enzyme activities and ion content were analyzed for a sensitive and a tolerant cultivar. Nodal cuttings of the tolerant cultivar, Kennebec, and the sensitive cultivar, Concord, were exposed to media without or with 30, 60, 90 or 120 mmol/L NaCl for 4 weeks. On exposure to NaCl, the length and fresh and dry weight of both shoots and roots of Concord showed greater decrease than those of Kennebec. The decrease in shoot growth was more severe than that of the root for both cultivars. The K+ content of shoots and roots of both cultivars was reduced in a dose-dependent manner by exposure to NaCl; the Na+ content Increased. Activities of ascorbate peroxidase, catalase and glutathione reductase were increased in NaCl-exposed shoots of Kennebec; the corresponding activities inNaCl-exposed shoots of Concord were decreased. Roots of both cultivars showed similar changes in the activities of these enzymes on exposure to NaCl. These studies established that enzyme activities In Concord shoots are inversely related to the NaCl concentration, whereas those in Kennebec do not show a dose dependency, which is also the case for the roots of both cultivars. Our findings suggest that an Increase in activity of antioxidant enzymes, such as ascorbate peroxidase,cetalase and glutathione reductase, can contribute to salt tolerance in Kennebec, a salt resistant cultivar of potato.