Heat shock induced proteins in plant cells

Tobacco (Nicotiana tabacum) and soybean (Glycine max) tissue culture cells were exposed to a heat shock and protein synthesis studied by SDS-polyacrylamide gel electrophoresis after labeling with radioactive amino acids. A new pattern of protein synthesis is observed in heat-shocked cells compared to that in control cells. About 12 protein bands, some newly appearing, others synthesized in greatly increased quantities in heat-shock cells, are seen. Several of the heat-shock proteins (HSPs) in both tobacco and soybean are similar in size. One of the HSPs in soybean (76K) shares peptide homology with its presumptive 25°C counterpart, indicating that the synthesis of at least some HSPs may not be due to activation of new genes. The optimum temperature for maximal induction of most HSPs is 39–40°C. Total protein synthesis decreases as heat-shock temperature is increased and is barely detectable at 45°C. The heat-shock response is maintained for a relatively short time in tobacco cells. After 3 hr at 39°C, a decrease is seen in the synthesis of the HSPs, and after 4 hr practically no HSPs are synthesized. After exposure to 39°C for 1 hr, followed by a return of tobacco cells to 26°C, recovery to the control pattern of synthesis requires greater than 6 hours. These results indicate that cells of flowering plants exhibit a heat-shock response similar to that observed in animal cells.     

Hormonal mechanisms for differentiation in plant tissue culture

Summary Cells possess extraordinary powers to organize their molecular processes not only to maintain a cell in a given steady state but also to recognize that state during differentiation. Regulation of these organizational forces appears to be under the control of chemical factors, and a hormonal concept of regulation has evolved. Hormones have been considered to act by reacting with a specific target site. This may be part of their mode of action, but I would like to suggest that a hormone enters and becomes part of a total molecular resonance system. In so doing, the entire molecular system of the cell is modified. Of the known plant hormones, the cytokinins, because of their role in experimentally induced cell division and differentiation, serve as a probe of hormonal involvement in differentiation. Cultured somatic cells of tobacco plants can be induced to undergo differentiation by addition of cytokinin and auxin to the medium. Studies of the cytokinin hormones show a series of diverse molecular involvements. The archetype cytokinin, N⁶-(Δ²-isopentenyl) adenosine (i⁶Ado), occurs in some molecular species of tRNA where it plays a vital role in the codon-anticodon interaction of tRNA and m-RNA. i⁶Ado under-goes extensive metabolism in the tobacco tissue. It is either degraded to adenosine or converted to derivatives that possess biological activity. It is perhaps, therefore, more correct to consider the hormone function as being derived from this total metabolic web. The normal somatic cells of tobacco cultures spontaneously change occasionally into an autonomous form that requires no external growth factors. This line of cells synthesizes i⁶Ado. The metabolic web of the hormone-dependent strain can be perturbed by added auxin but such is not the case in the autonomous strain. These data provide some insight into the altered state of cytokinin activity in which a cell line changes into an autonomous form. Curiously, in become independent of the requirement for exogenous cytokinin, the autonomous tissue becomes sensitive to added cytokinin. i⁶Ado also inhibits the growth of lines of mammalian cancer cells grown in culture. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Isoperoxidases from tobacco tissue cultures

Two anodic isoperoxidases (A₁ and A₂) from tobacco tissue culture W-38 and two cathodic isoperoxidases (C₃ and C₄) from tobacco suspension culture WR-132 have been separated and characterized. Molecular weights for each of the isoperoxidases have been determined by two different methods. Only C₄ contained a carbohydrate component. The substrate specificity and the pH optima for the four enzymes with each of five substrates were determined.     

Preparation of an isomorphous heavy-atom derivative of tobacco mosaic virus by chemical modification with 4-sulpho-phenylisothiocyanate

The reaction of the vulgare and U2 strains of tobacco mosaic virus with 4-sulpho-phenylisothiocyanate has been investigated. The coat protein of the U2 strain has a proline residue at its N-terminus and a lysine residue at position 53. Whereas both residues could be reacted with 4-sulpho-phenylisothiocyanate in the isolated coat protein, only proline-1 was modified during treatment of the intact virus with the same reagent, thereby showing that the loss of reactivity of the ?-amino group of lysine-53 is a consequence of the virus structure. The 4-sulpho-phenylthiocarbamoyl derivative of amino groups shows considerable tautomerism and, as a consequence, it proved possible to prepare a heavy-atom derivative of the intact U2 strain in which methyl mercury nitrate was bound by the modified N-terminal residue of the coat protein.On the other hand, when the intact vulgare strain was treated with 4-sulphophenylisothiocyanate, little or no modification of the ?-amino groups of the two lysine residues (positions 53 and 68) per polypeptide chain was observed. Taking into account previous studies on the reactivity of the amino groups of the coat protein in tobacco mosaic virus vulgare and assuming that all strains and mutants have closely similar three-dimensional structures, these experiments suggest that the N-terminal residue is more exposed (i.e. probably nearer the virus “surface”) than the side-chain of lysine-68, which in turn is more accessible than the side-chain of lysine-53. This interpretation is readily compatible with the results of X-ray diffraction analysis carried out on these chemically modified viruses (Mandelkow &; Holmes, 1974) and lends support to the hope that such methods of preparing heavy-atom derivatives of proteins will be of general use.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Spin-lattice Relaxation in the Proton NMR of Hydrated Tobacco Cut-fillers

The spin-lattice relaxation time was measured by proton NMR of hydrated tobacco cut-fillers. The relaxation decays of adsorbed water were expressed by a single phase system below 70% relative humidity, while a two-phase system was applicable to water adsorbed at more than 80% relative humidity. From the two-phase model, it was considered that 0.12–0.13 kg water/kg dry tobacco is bound water.     

白腐真菌血红密孔菌漆酶复合酶预处理烟梗丝的响应面法优化

烟梗是烟草工业的重要副产物,也是宝贵的自然资源。本研究首先利用白腐菌漆酶对烟梗丝进行预处理,提升了添加烟梗丝的卷烟品质;然后分别以木质素、纤维素、半纤维素和果胶的降解率为响应值,采用Box-Behnken设计建立方程模型,对漆酶、纤维素酶、半纤维素酶和果胶酶组成的复合酶预处理烟梗丝条件进行了优化。结果表明：每100g烟梗丝加入30U漆酶,在料液比为35%、温度为30℃、酶解pH为5处理48h的条件下预处理的烟梗丝对提升卷烟品吸效果最佳,烟梗丝中木质素、纤维素、半纤维素和果胶的降解率分别为20.16%、15.10%、7.20%和12.40%;为获得与之相同的各组分降解率,响应面法优化漆酶复合酶最佳处理条件为：每100g烟梗丝加入漆酶14.72U、纤维素酶1.00U、半纤维素酶1.00U、果胶酶8.45U。验证发现烟梗丝各组分降解率实测值与理论值无显著性差异,且显微结构观察显示复合酶处理后的烟梗丝表面致密结构被破坏,孔洞数量明显增加。本研究获得的白腐菌漆酶预处理后的烟梗丝在卷烟中的添加能有效改善卷烟品质,且漆酶复合酶的使用大幅减少了漆酶的用量,降低了漆酶预处理烟梗丝的成本,为废弃烟梗生物质的资源化利用提供了重要依据。     

小黑杨PsnHB13基因在烟草中的遗传转化与功能分析

克隆小黑杨HD-ZIP家族基因PsnHB13,对该基因进行生物信息学分析、过表达载体构建、烟草的遗传转化。结果表明小黑杨PsnHB13基因cDNA全长870 bp,编码289个氨基酸。成功构建植物过表达载体pROKⅡ-PsnHB13,并通过农杆菌介导的叶盘法将外源基因转入野生型烟草。检测结果显示PsnHB13已成功整合入烟草基因组中,并在mRNA水平表达。通过观察转基因烟草的生长,发现过表达PsnHB13基因的烟草与野生型相比出现叶面积变小、叶型变长,根系生长缓慢,花朵变小等明显表型。说明PsnHB13基因主要对烟草叶片、根系及花的生长发育起到负调控作用。本文为研究PsnHB13基因对杨树生长发育的影响提供理论基础。     

A green and effective method for the determination of metalaxyl enantiomers in tobacco and soil by supercritical fluid chromatography–tandem mass spectrometry

We reported a new methodology for the stereoselective determination of metalaxyl enantiomers in tobacco and soil. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was used for the extraction and clean-up of the tobacco and soil samples. Separation of the metalaxyl enantiomers was performed on an ACQUITY UPC2 Trefoil CEL1 chiral column coupled with supercritical fluid chromatography with tandem mass spectrometry (SFC-MS/MS), and the run time was only 5 minutes. Under the optimized conditions, the recoveries for the enantiomers were between 78.2% and 93.3% with intraday relative standard deviations (RSDs) ranging from 1.1% to 5.4%. The limit of detection (LOD) for the enantiomers in tobacco and soil varied from 0.005 to 0.007 mg/kg, and the limit of quantitation (LOQ) ranged from 0.017 to 0.020 mg/kg. In this method, only a small amount of methanol was consumed to obtain a rapid stereoselective separation. This proposed method showed good accuracy and precision and might be suitable for fast enantioselective determination of metalaxyl in food and environmental samples. The developed method was further validated by application to the analysis of authentic samples.