Infection, propagation, distribution and stability of plant virus in hairy root cultures

Nicotiana benthamiana hairy root cultures were infected with tobacco mosaic virus (TMV) and used for in vitro plant virus propagation. The roots were infected with TMV by addition of virus to the medium at the same time as root inoculation. Viral accumulation in the biomass was 7-11-fold greater when the initial infection was carried out in B5 medium rather than sodium phosphate buffer; virus accumulation also increased with increasing viral inoculum concentration. The amount of TMV accumulated in the biomass was similar when virus was retained in the medium for the duration of the cultures and when the inoculum virus was removed 23h after addition to the roots. In roots with established infections, the concentration of virus remained relatively constant and did not increase with further root growth. The distribution of virus within individual root mats harvested from shake flasks was not uniform; there was also significant variability in viral accumulation between replicate hairy root cultures. The picture that emerges from this work is that in vitro viral accumulation in hairy root cultures depends strongly on the viral inoculum concentration applied and the initial level of primary infection achieved, even though primary infection by external virus occurs mainly within only the first few hours of exposure to the biomass and is followed by substantial secondary infection by viral progeny within the root tissue.     

Artificial seeds for plant propagation

Artificial seeds consisting of tissue-culture produced embryos encased in a protective coating, will allow the economical mass propagation of elite plant varieties. They would also be a channel for new plant lines produced through biotechnological advances to be delivered directly to the greenhouse or field.     

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.     

In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation

Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82 ± 0.14 mg g⁻¹ dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80–90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 μg g⁻¹ dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.     

Compartmentation in plant metabolism

Cell fractionation and immunohistochemical studies in the last 40 years have revealed the extensive compartmentation of plant metabolism. In recent years, new protein mass spectrometry and fluorescent-protein tagging technologies have accelerated the flow of information, especially for Arabidopsis thaliana, but the intracellular locations of the majority of proteins in the plant proteome are still not known. Prediction programs that search for targeting information within protein sequences can be applied to whole proteomes, but predictions from different programs often do not agree with each other or, indeed, with experimentally determined results. The compartmentation of most pathways of primary metabolism is generally covered in plant physiology textbooks, so the focus here is mainly on newly discovered metabolic pathways in plants or pathways that have recently been revised. Ultimately, all of the pathways of plant metabolism are interconnected, and a major challenge facing plant biochemists is to understand the regulation and control of metabolic networks. One of the best-characterized networks links sucrose synthesis in the cytosol with photosynthetic CO(2) fixation and starch synthesis in the chloroplasts. One of the key features of this network is how the transport of pathway intermediates and signal metabolites across the chloroplast envelope conveys information between the two compartments, influencing the regulation of several enzymes to co-ordinate fluxes through the different pathways. It is widely accepted that chloroplasts and mitochondria originated from prokaryotic endosymbionts, and that new transporters and regulatory networks evolved to integrate metabolism in these organelles with the rest of the cell. Curiously, the present-day locations of many metabolic pathways within the cell often do not reflect their evolutionary origin, and there is evidence of extensive shuffling of enzymes and whole pathways between compartments during the evolution of plants.     

NMR and plant metabolism

Recent advances in NMR methodology offer a way to acquire a comprehensive profile of a wide range of metabolites from various plant tissues or cells. NMR is a powerful approach for plant metabolite profiling and provides a capacity for the dynamic exploration of plant metabolism that is virtually unmatched by any other analytical technique.     

Resolving subcellular plant metabolism

Plant cells are characterized by a high degree of compartmentalization and a diverse proteome and metabolome. Only a very limited number of studies has addressed combined subcellular proteomics and metabolomics which strongly limits biochemical and physiological interpretation of large‐scale ’omics data. Our study presents a methodological combination of nonaqueous fractionation, shotgun proteomics, enzyme activities and metabolomics to reveal subcellular diurnal dynamics of plant metabolism. Subcellular marker protein sets were identified and enzymatically validated to resolve metabolism in a four‐compartment model comprising chloroplasts, cytosol, vacuole and mitochondria. These marker sets are now available for future studies that aim to monitor subcellular metabolome and proteome dynamics. Comparing subcellular dynamics in wild type plants and HXK1‐deficient gin2‐1 mutants revealed a strong impact of HXK1 activity on metabolome dynamics in multiple compartments. Glucose accumulation in the cytosol of gin2‐1 was accompanied by diminished vacuolar glucose levels. Subcellular dynamics of pyruvate, succinate and fumarate amounts were significantly affected in gin2‐1 and coincided with differential mitochondrial proteome dynamics. Lowered mitochondrial glycine and serine amounts in gin2‐1 together with reduced abundance of photorespiratory proteins indicated an effect of the gin2‐1 mutation on photorespiratory capacity. Our findings highlight the necessity to resolve plant metabolism to a subcellular level to provide a causal relationship between metabolites, proteins and metabolic pathway regulation.     

Hydroxycinnamoyltransferases in plant metabolism

Hydroxycinnamoyltransferases are enzymes transferring hydroxycinnamoyl units like cinnamoyl, 4-coumaroyl, caffeoyl, feruloyl and sinapoyl moieties from an activating residue such as coenzyme A or glucose or activated as hydroxycinnamoyl ester (e.g. chlorogenate) to an acceptor molecule, most commonly to an OH or NH₂ group as ester or amide. The hydroxycinnamoyl groups play either a “decorating” role or are building blocks of more complex structures. Proteins catalysing hydroxycinnamoyl transfer have been known for many decades and are nowadays investigated on molecular and structural levels. At least four different protein families give rise to enzymes with hydroxycinnamoyltransferase activity: serine carboxypeptidase-like proteins, tyramine hydroxycinnamoyltransferase-like enzymes, BAHD acyltransferases and GDSL-lipase/esterase-like enzymes. Interestingly, the same or very similar products can be formed by enzymes from different enzyme classes and using differently activated hydroxycinnamoyl units. This review will summarise the current literature data on the features of hydroxycinnamoyltransferases from the four different enzyme groups.     

c-Fos regulates hepatitis C virus propagation

Hepatitis C virus (HCV) RNA replication requires cellular factors as well as viral non-structural proteins (NS protein). Using small interfering RNA (siRNA) library screening, we previously identified c-Fos as a host factor involved in HCV propagation. In the present study, we demonstrated that silencing of c-Fos expression resulted in decrease of HCV propagation in cell culture grown HCV (HCVcc)-infected cells; whereas overexpression of c-Fos significantly increased HCV propagation. We further confirmed the positive role of c-Fos in HCV propagation by both HCV-luciferase reporter assay and immunofluorescence analysis. We showed that c-Fos level was upregulated by HCV infection. Furthermore, phorbol 12-myristate 13-acetate (PMA)-induced c-Fos level was synergistically increased by HCV infection. These data suggest that c-Fos acts as a positive regulator of HCV propagation and may contribute to HCV-associated pathogenesis.     

Dark starvation and plant metabolism

Summary Photooxidation of hydroxylamine to nitrite by spinach (Spinacia oleracea L. and sugarbeet (Beta vulgaris L.) chloroplast lamellae in the presence of autoxidable electron acceptors is inhibited by either solubilized or membrane-bound superoxide dismutase (SOD). This inhibition is reversed by KCN. The rates of hydroxylamine photooxidation by chloroplast lamellae, a reaction which is apparently driven by the superoxide free-radical ion, was used for quantitating the amount of SOD bound to chloroplast lamellae, as compared to a soluble enzyme of defined concentration. After digitonin fragmentation of chloroplast lamellae, ca. 80% of the SOD activity is associated with subchloroplast particles sedimenting after 2 h centrifugation at 200 000 x g. Less than 10% of the SOD activity is associated with particles sedimenting after centrifugation for 30 min at 20 000 x g. 5–10% of the cyanide-sensitive SOD is recovered in the soluble fraction of the subchloroplast-free supernatant after centrifugation at 200 000 x g for 2 h.Abbreviation SOD superoxide dismutase     

Regulation of plant glucosinolate metabolism

Glucosinolates and their degradation products are known to play important roles in plant interaction with herbivores and micro-organisms. In addition, they are important for human life. For example, some degradation products are flavor compounds and some exhibit anticarcinogenic properties. Recent years have seen great progress made in the understanding of glucosinolate biosynthesis in Arabidopsis thaliana. The core glucosinolate biosynthetic pathway has been revealed using biochemical and reverse genetics approaches. Future research needs to focus on questions related to regulation and control of glucosinolate metabolism. Here we review current status of studies on the regulation of glucosinolate metabolism at different levels, and highlight future research towards elucidating the signaling and metabolic network that control glucosinolate metabolism.     

Dark starvation and plant metabolism

Summary The fixation pattern of radioactive labelled photosynthetic intermediates was followed under steady state conditions during prolonged dark starvation of spinach plants (Spinacia oleracea L.). It is suggested that the considerable increase of radioactive dihydroxyacetonephosphate is correlated with a specific leakage of the outer chloroplast envelope induced by dark starvation. The primary fixation product, phosphoglyceric acid, followed the same decreasing tendency as observed for the net CO₂ fixation. In contrast, the relative label in other intermediates is the same as in the controls. When after several days of dark starvation the plants were again transferred into light, a regeneration of the CO₂ fixation accompanied by the appearance of a normal fixation pattern was observed. Since the regeneration was prevented by the addition of lincomycin, the net increase is considered to be due to a new protein synthesis rather than a reactivation.Abbreviations GAPDH glyceraldehyde phosphate dehydrogenase (E.C. 1.2.1.13) - DHAP dihydroxyacetonphosphate - FDP fructose 1,6-diphosphate - glol glycolic acid - PGA 3-phosphoglyceric acid - S-D-P sugar diphosphates - S-M-P sugar monophosphates Part I: Postius and Jacobi (1971).     

Glial metabolism of quercetin reduces its neurotoxic potential

The neuroprotective effects of flavonoids will ultimately depend on their interaction with both neuronal and glial cells. In this study, we show that the potential neurotoxic effects of quercetin are modified by glial cell interactions. Specifically, quercetin is rapidly conjugated to glutathione within glial cells to yield 2′-glutathionyl-quercetin, which is exported from cells but has significantly reduced neurotoxicity. In addition, quercetin underwent intracellular O-methylation to yield 3′-O-methyl-quercetin and 4′-O-methyl-quercetin, although these were not exported from glia at the same rate as the glutathionyl adduct. The neurotoxic potential of both quercetin and 2′-glutathionyl-quercetin paralleled their ability to modulate the pro-survival Akt/PKB and extracellular signal-regulated kinase (ERK) signalling pathways. These data were supported by co-culture investigation, where the neurotoxic effects of quercetin were significantly reduced when they were cultured alongside glial cells. We propose that glial cells act to protect neurons against the neurotoxic effects of quercetin and that 2′-glutathionyl-quercetin represents a novel quercetin metabolite.     

药用植物大戟的快速繁殖研究

以野生大戟为材料,探讨了大戟茎尖扦插繁殖和组织培养等快速繁殖技术的条件。结果表明:大戟茎 尖扦插繁殖,其成活率可以达到88.6%,用含有腋芽的茎在MS培养基上培养,发芽比例可以达到55%;用愈 伤组织诱导生芽,最高可以达到12%。嫩芽在不含激素的1/2MS培养基中培养,生根率达到47.1%。幼苗 接种本源的或同科外源植物5株内生真菌,比较其生长表明,接种大戟来源的两株内生真菌全苗重分别达到 对照的1.51和2.08倍,根重达到对照的2.09和3.68倍,最有利于宿主生长。