In vitro assembly of microtubules from tubulins of several higher plants

Tubulins were isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4B) and ion exchange (DEAE-Sephacel) chromatography from several higher plants (mung bean, pea, whole pod bean, zucchini, cucumber seedlings and carrot suspension cultured cells). All these higher plant tubulins readily polymerized to microtubules in a polymerization medium containing GTP, Mg2+, EGTA, leupeptin and DMSO. Tubulins from mung bean, pea and whole pod bean showed identical behaviour on polyacrylamide gel electrophoresis but differed from carrot zucchini and cucumber tubulin. Consequently, tubulin of higher plants seems to have different molecular properties in different plant species.     

3-Dehydroshikimate dehydratase in mung bean cultured cells

Summary The activity of 3-dehydroshikimate dehydratase was detected in an extract prepared from cells of mung bean (Vigna mungo) that had been cultured in the presence of shikimate while such activity was not detectable in an extract prepared from cells cultured without shikimate. The enzyme was partially purified and characterized. The maximum activity of the enzyme was observed at pH 7.4. The activity was inhibited to a small extent by EDTA and sulfhydryl inhibitors. The partially purified enzyme was sensitive to thermal denaturation but was stabilized by Mg²⁺ ions. These results suggest that 3-dehydroshikimate dehydratase might be induced in mung bean cultured cells in the presence of shikimic acid.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - DHS 3-dehydroshikimic acid - PCA protocatechuic acid - QA quinic acid - SA shikimic acid - SORase shikimate - NAEP oxidoreductase     

Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and methyljasmonate

In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with cyclodextrins alone is far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1,447.8 ± 60.4 μmol trans-resveratrol g⁻¹ dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 μmol trans-resveratrol g⁻¹ dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium with the maximal levels observed with 20 g l⁻¹ sucrose and the joint action of cyclodextrins and 100 μM methyljasmonate. The sucrose concentration did not seem to limit the process although it affects significantly the specific productivity since the lowest sucrose concentration is 10 g l⁻¹, the highest productivity is reached (100.7 ± 5.8 μmol trans-resveratrol g⁻¹ dry weight g⁻¹ sucrose) using cyclodextrins and 25 μM methyljasmonate.     

Suppression of the Defence-Related Oxidative Burst in Bean Leaf Tissue and Bean Suspension Cells by the Necrotrophic Pathogen Botrytis cinerea

The interaction of two selected isolates of Botrytis cinerea with bean suspension cells and bean leaf discs was compared in relation to levels of reactive oxygen intermediates (ROI). Isolate B 1.7 was arrested by a hypersensitive‐like necrosis of bean leaf tissue. According to its inability to spread and produce conidia on the bean leaf tissue it was classified as non‐aggressive. The second isolate induced a fast expanding light brownish necrosis of the leaf tissue. It was able to produce conidia on bean leaf discs and was classified as aggressive. The generation of superoxide was followed biochemically in inoculated bean cell suspensions. Both isolates induced a similar early superoxide peak approximately 18‐h post inoculation (hpi). While the non‐aggressive isolate induced a much stronger secondary superoxide burst at 33 hpi, the level of superoxide of suspension cells inoculated with the aggressive isolate was below the control level. This is the first report on the occurrence of a biphasic oxidative burst in plant cells induced by a fungal pathogen. Such a suppression of superoxide generation was also observed in bean leaf discs inoculated with the aggressive isolate. An oxidative burst‐suppressing agent was extracted from inoculated cell culture medium and determined as 2‐methyl‐succinate (2‐MS) by GC/MS analysis. The compound was detected approximately 20 hpi in the aggressive fungus–plant interaction. 2‐MS was able to suppress the hypersensitive response‐like necrosis on leaf discs as well as the second superoxide burst in suspension cells when inoculated with the non‐aggressive isolate. The early superoxide burst at 18 hpi was not affected. The results confirm the important role of enhanced production of ROI in plant resistance reactions, also for a necrotrophlike B. cinerea.     

绿豆幼苗生长状态对环境羟基自由基水平的影响

为了解植物生长状态对环境羟基自由基水平的影响,研究了相同培养条件下不同生长状态的绿豆(Vigna radiata)幼苗对空气中的羟基自由基水平的影响。结果表明,正常生长的绿豆幼苗周围环境羟基自由基水平显著高于没有植物生长的环境,失活幼苗对周围环境羟基自由水平没有显著影响;渗透胁迫的绿豆幼苗对环境羟基自由基水平影响极显著,渗透胁迫程度不同其影响程度也有所不同;绿豆幼苗对环境羟基自由基水平的影响与其呼吸速率密切相关。这证明绿豆幼苗生长对环境羟基自由基水平有影响,且这种影响依赖于其生理代谢过程及生长状态。     

A Comparative Analysis of in vitro cellulose synthesis from cell-free extracts of mung bean (Vigna radiata,Fabaceae) And Cotton (Gossypium hirsutum,Malvaceae)

A comparison of cellulose synthesized in vitro from primary walls of etiolated mung bean (Vigna radiata) seedlings and secondary walls of cotton fibers (Gossypium hirsutum) was made by applying conditions found to be essential for in vitro cellulose I assembly from cotton (Kudlicka et al., 1995, Plant Physiology, vol. 107, pp. 111–123). Mung bean fractions including the plasma membrane (PM), the first solubilized fraction (SE₁), and the second solubilized fraction (SE₂), incorporated more radioactive UDP-Glc into the total product than the same fractions from secondary walls. A significant difference was found with the mild digitonin solubilized fraction (SE₁), which produced eight times more total product than the SE₁ fraction of cotton. However, the SE₁ fraction from cotton produced a larger quantity of cellulose (32.1%) than from mung bean (6.9%). Treatment of the in vitro product by acetic/nitric acid reagent (AN) for varying periods of time demonstrated that cellulose synthesized in vitro from mung bean was more easily degraded than cellulose from cotton fibers. This would suggest that cellulose I produced in vitro from the cotton SE₁ fraction may have a higher crystallinity and DP than cellulose I produced in vitro from mung bean. The fibrils of cellulose produced by the SE, fraction of mung bean were loosely associated and not arranged into a compact bundle as in case of cellulose I synthesized by the cotton SE₁ fraction. The electron diffraction patterns (ED) of both products show reflections characteristic for cellulose I. Products from the SE₂ fraction of mung bean and cotton reveal similarities with the cellulose II allomorph synthesized, as well as abundant β-1,3-glucan.     

铁核桃根与叶水浸提液对果园间作绿肥植物的化感作用及其生理机制

以西南地区具有代表性的16种绿肥植物为受体材料,采用培养皿药膜法研究了铁核桃(Juglans sigillata)根水浸提液对受体种子发芽率及幼苗鲜重、干重的化感效应;并进一步研究了铁核桃根、叶水浸提液和胡桃醌对化感效应存在明显差异的4种绿肥植物(绿豆、红三叶、白三叶、花生)种子萌发与幼苗生长以及抗氧化酶特性的影响,以筛选适宜中国西南地区核桃园种植的绿肥植物,探讨核桃根和凋落物对绿肥作物的化感作用机制。结果表明:(1)铁核桃根水浸提液对绿豆的发芽率没有影响,但对绿豆幼苗鲜重和干重有显著抑制作用,而对其他15种绿肥的发芽率和鲜重、干重均有抑制作用。(2)胡桃醌显著抑制绿豆种子萌发,而铁核桃根或叶水浸提液对绿豆种子萌发没有影响。(3)铁核桃根或叶水浸提液以及胡桃醌对绿肥植物幼苗生长的化感效应趋势一致,但核桃根或叶水浸提液的化感效应强于胡桃醌。(4)绿豆幼苗在铁核桃根或叶水浸提液以及胡桃醌处理下,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)的活性均高于其他3种(红三叶、白三叶、花生)受体幼苗,表明绿豆清除活性氧能力高,细胞受损害程度较低,受化感作用影响最弱。研究认为,绿豆为适宜中国西南地区幼龄核桃园种植的间作绿肥植物。     

Loss of adaptation following reversion suggests trade‐offs in host use by a seed beetle

Experimental evolution has provided little support for the hypothesis that the narrow diets of herbivorous insects reflect trade‐offs in performance across hosts; selection lines can sometimes adapt to an inferior novel host without a decline in performance on the ancestral host. An alternative approach for detecting trade‐offs would be to measure adaptation decay after selection is relaxed, that is, when populations newly adapted to a novel host are reverted to the ancestral one. Lines of the seed beetle Callosobruchus maculatus rapidly adapted to a poor host (lentil); survival in lentil seeds increased from 2% to > 90% in < 30 generations. After the lines had reached a plateau with respect to survival in lentil, sublines were reverted to the ancestral host, mung bean. Twelve generations of reversion had little effect on performance in lentil, but after 25–35 generations, the reverted lines exhibited lower survival, slower development and smaller size. The most divergent pair of lines was then assayed on both lentil and mung bean. Performance on lentil was again much poorer in the reverted line than in the nonreverted one, but the lines performed equally well on mung bean. Moreover, the performance of the nonreverted line on mung bean remained comparable to that of the original mung‐bean population. Our results thus present a paradox: loss of adaptation to lentil following reversion implies a trade‐off, but the continued strong performance of lentil‐adapted lines on mung bean does not. Genomic comparisons of the reverted, nonreverted and ancestral lines may resolve this paradox and determine the importance of selection vs. drift in causing a loss of adaptation following reversion.     

A Rapid Method for Evaluation of Autoxidation and Antioxidants

To examine the mechanism of starch degradation in legume cotyledons and the physiological role of α-glucosidase, mung bean seeds were germinated in the presence of Bay m 1099, an α-glucosidase inhibitor. Bay m 1099 (10 μg/ml medium), which minimized the growth deterioration of the mung bean seedlings, caused no changes in the overall rate of starch degradation and of soluble carbohydrate production in the cotyledons, although α-glucosidase activity had been completely suppressed. Total amylase and phosphorylase activities were not influenced by Bay m 1099. These results suggest that the mung bean α-glucosidase is less responsible for starch degradation, unlike wheat α-glucosidase [Konishi et al., Biosci. Biotech. Biochem., 58, 135-139 (1994)].     

Etiology and control of durian foliar blight and dieback caused by Rhizoctonia solani

Foliar blight and dieback of durian seedlings and trees in Peninsular Malaysia was found to be caused by Rhizoctonia solani (teleomorph - Thanatephoms cucumeris) The fungus grew well and produced abundant sclerotia at temperatures higher than 24°C with an optimum at 28°C. It grew poorly at 35°C and did not grow at 10°C. The strains studied were found to belong to the anastomosis group AG-1. They were pathogenic on durian, papaya, cucumber, long bean, Mikania weed, padi, musk melon, mung bean, Zoysia grass, Bermuda grass, and St Augustine grass. They were mildly pathogenic on groundnut, and non-pathogenic on maize, guava and Brassica‘pak choy’. The disease was effectively controlled by foliar sprays of pencycuron and benomyl; triadimefon and an antagonistic bacterium suspension treatment were less effective and quintozene-etridiazole mixture gave poor disease control.     

Fermentation of soybean oil deodorizer distillate with Candida tropicalis to concentrate phytosterols and to produce sterols-rich yeast cells

Phytosterols have been recovered from the deodorizer distillate produced in the final deodorization step of vegetable oil refining by various processes. The deodorizer distillate contains mainly free fatty acids (FFAs), phytosterols, and tocopherols. The presence of FFAs hinders recovery of phytosterols. In this study, fermentation of soybean oil deodorizer distillate (SODD) with Candida tropicalis 1253 was carried out. FFAs were utilized as carbon source and converted into cellular components as the yeast cells grew. Phytosterols concentration in SODD increased from 15.2 to 28.43 % after fermentation. No significant loss of phytosterols was observed during the process. Microbial fermentation of SODD is a potential approach to concentrate phytosterols before the recovery of phytosterols from SODD. During SODD fermentation, sterols-rich yeast cells were produced and the content of total sterols was as high as 6.96 %, but its major sterol was not ergosterol, which is the major sterol encountered in Saccharomyces cerevisiae. Except ergosterol, other sterols synthesized in the cells need to be identified.     

Metabolic Conversion of Castasterone and Brassinolide into Their Glucosides in Higher Plants

Castasterone (CS) and brassinolide (BL) were administered to mung bean (Vigna radiata) explants, Arabidopsis thaliana seedlings, and cultured Catharanthus roseus cells, and the glucosylated metabolites were analyzed using LC/MS/MS. In mung bean and C. roseus, CS-2-O-glucoside (CS-2G), -3-O-glucoside (CS-3G), -22-O-glucoside (CS-22G), and -23-O-glucoside (CS-23G) were identified as metabolites of CS, whereas BL-2G, BL-3G, and BL-23G were identified as metabolites of BL. In A. thaliana, CS and BL were converted into their respective 2-O- and 23-O-glucosides. Of the metabolites identified with BL and CS administration, BL-23G was the predominant metabolite in mung bean and A. thaliana, whereas the 3-O-glucoside of BL was abundant in C. roseus. This is the first report of the metabolic conversion of CS into CS-2G, CS-3G, CS-22G, and CS-23G, and of BL into BL-2G and BL-3G. Our results indicate that the glucosylation profiles of BL and CS vary with plant species, and that the glucosylation of CS is rather limited quantitatively, compared with that of BL.     

Studies of the Effect of Tenuazonic Acid on Plant Cells and Seedlings

The biological and biochemical studies of the effect of tenuazonic acid on plant cells and seedlings were carried out. Tenuazonic acid exhibited a conspicuous stunting effect on the seedling-growth of rice plant, mung bean, radish and turnip, and on the growth of suspension cultured cells of soybean and rice plants. Tenuazonic acid exhibited no effect on the O₂-uptake and the activity of SH-enzyme of the plant, but inhibited the incorporation of ¹⁴C-Ieucine into the protein fraction and that of ¹⁴C-adenine into nucleic acid fraction of suspension cultured soybean cells as well as these uptake into the cells. And then it has been proved that these incorporation-inhibitions were not merely due to the inhibition of ¹⁴C-leucine and ¹⁴C-adenine uptake into the cells but based on the intrinsic inhibition of protein and nucleic acid syntheses, respectively.     

Effects of 2-benzoxazolinone on the germination, early growth and morphogenetic response of mung bean (Phaseolus aureus)

2‐Benzoxazolinone (BOA), a type of hydroxamic acid present in cereals and implicated in allelopathy, is now being viewed as a potential candidate for the development of natural herbicides. A study was conducted to determine the effect of BOA on mung bean (Phaseolus aureus) through a multitude of bioassays to understand its physiological and biochemical action. It was observed that BOA significantly decreased the germination of mung bean and its early growth (measured in terms of seedling length and dry weight). A typical dose–response relationship was observed with BOA treatment, and I₅₀ values (concentrations at which 50% inhibition occurs) for germination, seedling length and seedling dry weight were calculated to be 4.3, 0.71 and 0.77 mM , respectively. There was therefore a greater inhibitory effect on seedling growth than on germination. Treated seedlings were characterised by a loss of chlorophyll and decreased respiratory activity, indicating a possible adverse effect of BOA on photosynthetic and respiratory metabolism. Mitotic activity in root‐tip cells of onion (Allium cepa) was completely arrested in response to BOA treatment, and the cells exhibited abnormality in shape and size. BOA also adversely affected rhizogenesis in hypocotyl cuttings of mung bean, indicating an impact on morphogenetic potential. It was associated with significant changes in the protein content and activities of proteases and polyphenol oxidases during the root development phase. This study concludes that BOA interfered with essential biochemical processes in mung bean. Such studies provide useful information on the biochemical and physiological modes of actions of BOA, with a view to its use as a herbicidal compound.     

Substrate-dependent auxin production by Rhizobium phaseoli improves the growth and yield of Vigna radiata L. under salt stress conditions

Rhizobium phaseoli strains were isolated from the mung bean nodules, and, the most salt tolerant and high auxin producing rhizobial isolate N20 was evaluated in the presence and absence of L-tryptophan (L-TRP) for improving growth and yield of mung bean under saline conditions in a pot experiment. Mung bean seeds were inoculated with peat-based inoculum and NP fertilizers were applied at 30-60 kg ha-1, respectively. Results revealed that imposition of salinity reduced the growth and yield of mung bean. On the contrary, separate application of L-TRP and rhizobium appeared to mitigate the adverse effects of salt stress. However, their combined application produced more pronounced effects and increased the plant height (28.2%), number of nodules plant-1 (71.4%), plant biomass (61.2%), grain yield (65.3%) and grain nitrogen concentration (22.4%) compared with untreated control. The growth promotion effect might be due to higher auxin production in the rhizosphere and improved mineral uptake that reduced adverse effects of salinity. The results imply that supplementing rhizobium inoculation with L-TRP could be a useful approach for improving growth and yield of mung bean under salt stressed conditions.     

Production of tocopherols by cell culture of safflower

A new method for the production of tocopherols by safflower (Carthamus tinctorius) cell culture has been developed. The main tocopherol produced is α-tocopherol which has the strongest vitamin E activity among tocopherol analogues. In a time course experiment tocopherol production showed a secondary metabolic pattern rather than a primary one. Better cell lines in terms of both growth rate and amounts of tocopherols produced were obtained by selection using various growth regulators and media additives. In addition, tocopherol production was effectively stimulated by administration of biosynthetic precursors. In particular, phytol increased the total tocopherol content by some 18-fold, i.e. 63.6 mg per 100 g dry weight, and the α-tocopherol content by some 11- fold i.e. 28.8 mg per 100 g dry weight.     

Production of pinoresinol diglucoside,pinoresinol monoglucoside,and pinoresinol by <Emphasis Type="Italic">Phomopsis</Emphasis> sp. XP-8 using mung bean and its major components

Phomopsis sp. XP-8 is an endophytic fungus that has the ability to produce pinoresinol diglucoside (PDG) in vitro and thus has potential application for the biosynthesis of PDG independent of plants. When cultivated in mung bean medium, PDG production was significantly improved and pinoresinol monoglucoside (PMG) and pinoresinol (Pin) were also found in the culture medium. In this experiment, starch, protein, and polysaccharides were isolated from mung beans and separately used as the sole substrate in order to explore the mechanism of fermentation and identify the major substrates that attributed to the biotransformation of PDG, PMG, and Pin. The production of PDG, PMG, and Pin was monitored using high-performance liquid chromatography (HPLC) and confirmed using HPLC-MS. Activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) were analyzed and tracked during the cultivation. The reaction system contained the compounds isolated from mung bean in the designed amount. Accumulation of phenylalanine, cinnamic acid, p-coumaric acid, PDG, PMG, and Pin and the activities of PAL, C4H, and 4CL were measured during the bioconversion. PMG was found only when mung bean polysaccharide was analyzed, while production of PDG and Pin were found when both polysaccharide and starch were analyzed. After examining the monosaccharide composition of the mung bean polysaccharide and the effect of the different monosaccharides had on the production of PMG, PDG, and Pin, galactose in mung bean polysaccharide proved to be the major factor that stimulates the production of PMG.