Two pathogenesis-related peroxidases in greengram (Vigna radiata (L.) wilczek) leaves and cultured cells induced by Macrophomina phaseolina (Tassi) Goid. and its elicitor

An elicitor has been isolated from Macrophomina phaseolina, the root rot and leaf blight pathogen of greengram. Suspension-cultured cells of greengram were established which responded to the fungal elicitor. When greengram leaves were inoculated with M. phaseolina two new peroxidases appeared. Similarly, two new peroxidases could be detected in suspension-cultured greengram cells when treated with the fungal elicitor. These peroxidases were purified by column chromatography and their molecular masses were 27 and 38 kDa. The new peroxidases detected in both leaves and cultured cells appear to be similar with the same molecular weights.     

Gene expression of ascorbic acid-related enzymes in tobacco

GDP-D-mannose pyrophosphorylase (GMPase) and L-galactono-1, 4-lactone dehydrogenase (GalLDH) are key enzymes in L-ascorbic acid (AsA) biosynthesis of plants, and a full-length cDNA for GMPase was isolated from tobacco using PCR. Additionally, expression of GMPase, GalLDH and other AsA-related enzymes was examined in tobacco tissues and cultured BY-2 cells, and the relationship between their expression patterns and AsA content is discussed. It was found that the expression of GalLDH and GMPase mRNAs was markedly suppressed by loading AsA, suggesting that AsA concentration in the cells may regulate AsA biosynthesis. Moreover, the expression of GMPase and GalLDH mRNAs in tobacco leaf also suggested that AsA biosynthesis may be induced by light.     

Effects of ribavirin and adenine arabinoside on tobacco mosaic virus in Nicotiana tabacum L. var Xanthi tissue cultures

Although both ribavirin (1-β-ribofuranosyl-1,2,4-triazole-3carboxamide) and adenine arabinoside inhibited the multiplication of tobacco mosaic virus (TMV) in mechanically inoculated leaf tissues, neither chemical inhibited virus multiplication in unorganized tobacco callus after in vitro inoculation. The adenine deaminase inhibitor, pentostatin, did not increase the activity of adenine arabinoside in cultured cells. Several different developmental conditions and media did not increase the ability of either chemical to eradicate the virus from tobacco tissue cultures. However, the virus was eradicated from TMV-infected callus when grown in the presence of combinations of ribavirin and adenine arabinoside in shoot inducing medium.     

Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucus carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.     

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine ( Vitis vinifera ) L. cv. Sultanina) and regenerating tobacco ( Nicotiana tabacum ) L. cv. Xanthi). Using [³⁵S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.     

Effects of ribavirin and adenine arabinoside on tobacco mosaic virus in Nicotiana tabacum L. var Xanthi tissue cultures

Although both ribavirin (1--ribofuranosyl-1,2,4-triazole-3carboxamide) and adenine arabinoside inhibited the multiplication of tobacco mosaic virus (TMV) in mechanically inoculated leaf tissues, neither chemical inhibited virus multiplication in unorganized tobacco callus after in vitro inoculation. The adenine deaminase inhibitor, pentostatin, did not increase the activity of adenine arabinoside in cultured cells. Several different developmental conditions and media did not increase the ability of either chemical to eradicate the virus from tobacco tissue cultures. However, the virus was eradicated from TMV-infected callus when grown in the presence of combinations of ribavirin and adenine arabinoside in shoot inducing medium.     

Photosynthetic Characteristics of Photoautotrophically Cultured Cells of Tobacco

The photosynthetic characteristics of photoautotrophically culturedcells of tobacco (Nicotiana tabacum cv. Samsun NN) as well asthose of photomixotrophically cultured cells and green leaveswere investigated. Analyses revealed that on a fresh weightbasis cultured tobacco cells had lower chlorophyll contentsthan cells of green leaves. The chlorophyll content per chloro-plast,however, was almost the same in both types of cell, and thechloroplast number per cell accounted for only small differencesin the cellular chlorophyll content. This indicates that thelarger cell volume of cultured cells is the main factor in thedifference in the chlorophyll content of these cells. Photosynthetic activity measurements also showed differencesin the chloroplasts of cultured and leaf cells. The maximumactivities of photosystem I and the Hill reaction for the culturedcells were about half those for leaf cells on a per unit chlorophyllbasis. Moreover, photo-autotrophic cells had relatively constantphotosystem I and Hill reaction activities during growth; whereas,on a fresh weight basis these activities in leaf cells reflecteddevelopmental changes in the chlorophyll content. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis showedqualitatively similar thylakoid polypeptide compositions forcultured and leaf cells at all stages of growth even thoughthere were quantitative decreases in the contents of severalpolypeptides in the cultured green cells (especially in photomixotrophiccells) in comparison to the polypeptide contents of tobaccoleaves. We speculate that the lower photosynthetic activityof the cultured cells may be caused by this reduction in thecontents of certain thylakoid polypeptides. (Received November 14, 1988; Accepted June 19, 1989)     

A cytokinin mutant derived from cultured tobacco cells

Tissues cultured from the leaf lamina of wild-type Nicotiana tabacum L. cv. "Havana 425" plants require an exogenous source of cytokinin for rapid growth. In contrast, leaf tissues of plants heterozygous or homozygous for the partially dominant, monogenic habituated leaf (H1-1) trait, exhibit a cytokinin-autotrophic phenotype in culture. Here we show that the H1 trait can arise in culture. Cytokinin autotrophic variants were obtained by culturing wild-type tissues of leaf lamina successively on media containing reduced concentrations of the cytokinin, kinetin. Plants regenerated from clones of these variants exhibited the H1 phenotype, which segregated in breeding tests as expected for a dominant, monogenic trait. This trait, designated H1-2, is inherited at a different locus than the H1-1 trait described earlier. Our results show that cytokinin mutants can arise in cell culture and that at least two genes regulate the cytokinin requirement of cultured tobacco tissues.     

Histo-Cytological Observations on Callus and Bud Formation in Cultures of Nicotiana tabacum L.

Leaf explants of tobacco were cultured on MS medium supplemented with 2 mg/ l NAA and 0.5 mg/l BA for induction of callus formation, or supplemented with 2 mg/l BA for bud formation. Histocytological observations on callus and bud formation were carried out. Three days after cultivation, mesophyll cells enlarged, the nuclei became more apparent and dark stained, and starch accumulated in the cells. Cell divisions began in the mesophyll cells at the cut ends, in the palisade cells near the vascular bundles and in the vascular parenchyma. Mitotic activity then spreaded over tbc explants, and was most active at the edges of leaf explants. Regular rows of cells appeared as a result of series of transverse divisions in the palisade. The number of chloroplast in the mesophyll cells decreased and degenerated gradually. A number of meristemoids ware initiated in the cultured leaf explants after 7 days of cultivation. They were originated from two kinds of tissues, the mesophyll and vascular bundle, including the phloem parenchyma and vascular sheath. On the medium with NAA and BA, callus formation was induced with vigorous divisions, whereas bud primordia were differentiated from the meristomoids on the medimn with 2 mg/l BA. The buds were developed from both the superficial meristemoids and the meristematic regions deep within the callused leaf explants. The accumulated starch in the cells gradually disappeared as bud formation proceeded.     

Constitutive expression of the neutral PR-5 (OLP, PR-5d) gene in roots and cultured cells of tobacco is mediated by ethylene-responsive cis-element AGCCGCC sequences

The constitutive accumulation of tobacco neutral PR-5 (osmotin-like protein; OLP, PR-5d) in roots and cultured cells was studied in transgenic tobacco plants harboring the OLP promoter::GUS gene. This construct showed strong β-glucuronidase expression in vascular tissues and cortex of roots as well as in cultured cells. Analysis using a mutated promoter showed that ethylene-responsive elements (AGCCGCC) were necessary for constitutive expression in roots and cultured cells. An electrophoretic mobility shift assay indicated that ERF3 (EREBP3), an ethylene-responsive-element-binding factor that was reported to be expressed in roots and in cultured cells as well as in ethephon-treated leaves, could bind to the AGCCGCC sequences of the OLP gene. These findings suggest that AGCCGCC sequences and ERFs mediate the constitutive expression of the OLP gene in roots and cultured cells of tobacco. Received: 14 November 1997 / Revision received: 29 May 1998 / Accepted: 8 July 1998     

Evaluation of 12 β-lactam antibiotics for Agrobacterium-mediated transformation through in planta antibacterial activities and phytotoxicities

The antibacterial activities of 12 beta-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 living in tobacco (Nicotiana tabacum L.) leaf tissues, and their phytotoxicities to tobacco leaf tissues were evaluated. All beta-lactams at minimum bactericidal concentration (MBC) or higher showed weak bactericidal activities against agrobacteria persisting in tobacco leaf tissues. The beta-lactams evaluated were classified into two major groups according to their inhibitory effect on shoot regeneration of tobacco leaf tissues: (1) highly phytotoxic drugs, and (2) moderately phytotoxic drugs. According to these results, suitable kind and concentration of beta-lactam antibiotics were evaluated for Agrobacterium-mediated transformation.     

Fine Structure of Healthy and TMV-Infected Tobacco Cells Grown in Tissue Culture

Three types of tobacco (Nicotiana tabacum cv. Havana 38) callus: 1) healthy stem callus, 2) TMV-infected stem callus, 3) TMV-infected leaf callus; and leaves differentiated from healthy stem callus, and from TMV-infected leaf callus were compared for fine structure. In addition, the fine structure was observed of plastids in cells of leaves differentiated from callus isolated from stem sections of TMV-infected hybrid tobacco plants (N. tabacum cv. Havana 38 ×N. glutinosa) grown under high temperature. The cytoplasmic organelles in tissue cultured cells were similar to those in cells of greenhouse-grown tobacco plants. Except for plastids, TMV infection did not noticeably affect morphologically other cellular organelles in tissue culture cells. In TMV-infected leaf callus, numerous small bodies were seen in plastid-like bodies, while vesicle-like structures were observed in the stroma of plastids in leaves differentiated from callus of hybrid tobacco inoculated with TMV. Morphological variations of mitochondria, such as swelling and vacuolization of the inner matrix, occurred frequently in TMV-infected leaf callus. Needle-like crystalline inclusions or looped inclusions composed of many fine, long filaments were considered TMV particles orientated parallel to each other. The TMV particles were detected in the cytoplasm of tissue culture cells.     

De-novo synthesis and release of peroxidases in cell suspension cultures of Nicotiana tabacum L.

De-novo synthesis of acid and basic peroxidases has been studied in cell suspension cultures of tobacco by incorporation of ³H- and ¹⁴C-amino acids. Incorporation rates were found to be high for acid peroxidases and low for basic peroxidases. Synthesis of all peroxidases was inhibited by cycloheximide and actinomycin D. Subculturing of the cells increased the rates of radioactive amino-acid incorporation into all peroxidases within the first 24 h. This rise in peroxidase synthesis was correlated with the age of the transferred cells. The older the cells were the more pronounced was the effect. During the culture cycle the high rates of peroxidase synthesis at the second day dropped back to initial values. Peroxidase synthesis was thus inversely related to peroxidase accumulation which was very low at the beginning and increased continuously. By pulse-chase experiments it has been shown that newly synthesized acid peroxidases accumulated in the medium. This process was inhibited by monensin. Only the acid peroxidases were secreted into the cell wall and from there released. The basic peroxidases were not detectable in the medium.Abbreviations AA^* radioactive amino-acid mixture - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Incompatible pathogen infection results in enhanced reactive oxygen and cell death responses in transgenic tobacco expressing a hyperactive mutant calmodulin

Transgenic tobacco (Nicotiana tabacum L. cv. Wisconsin 38) lines expressing a mutant calmodulin (VU-3) that hyperactivates NAD kinase exhibit an enhanced elicitor-stimulated oxidative-burst reaction (S.A. Harding et al., 1997, EMBO J. 16: 1137–1144). VU-3 transgenic tobacco was used in the present study to investigate the relationship between calmodulin signalling, the production of active oxygen species and cell death in response to infection with an incompatible pathogen. Following P. syringae pv. syringae 61 infection, suspension cells derived from VU-3 transgenic plants exhibited a stronger oxidative burst (3- to 4-fold higher primary and secondary burst reactions), greater media alkalinization (3-fold) and more rapid cell death (4-fold greater mortality at 20 h post infection) than did infected control tobacco cells. Infection of leaf tissues with P. syringae pv. syringae 61 also resulted in an enhanced cell death response compared to control tobacco tissues. This cell death response of VU-3 leaf tissues, but not control leaf tissues, was further enhanced by the presence of 50 μM salicylic acid, suggesting that this transgenic line is more sensitive to the effects of this agent. Overall, the data support the model that calmodulin signalling pathways are involved in the plant oxidative burst and contribute to the regulation of cell death in infected plant tissues undergoing the hypersensitive response. Received: 6 January 1998 / Accepted: 7 March 1998     

Integrated regulation of the photosynthetic gene family from Arabidopsis thaliana in transformed tobacco cells

Summary Expression of the three chlorophyll a/b binding protein (cab) genes of Arabidopsis thaliana was studied in transformed tobacco tissues. For each cab gene, approximately 1000 bp of the promoter region plus a portion of the structural gene was inserted into a promoter-expression vector such that a translational fusion between the cab gene and the promoter-less chloramphenicol acetyltransferase (cat) gene was formed. The constructed molecules were introduced into either cultured tobacco cells or tobacco leaves and the promoter activity was monitored as chloramphenicol acetyltransferase activity. The light-grown tissues exhibited 1.5- to 60-fold greater promoter activity than did dark-grown tissues. Expression of the cab promoters was tissue specific: activities were much stronger in green leaves than other tissues. The cab promoters were almost equally active in transformed calli or shoots derived from leaves. However, in cultured tobacco cells, one promoter was two to three times stronger than the other two. The chimeric gene fusion, cab-cat, segregated in the F1 generation as a dominant Mendelian trait.     

烟草叶片愈伤组织形成过程中的细胞程序性死亡

以烟草叶片在MS培养基上诱导的10个不同时期的愈伤组织为材料,通过荧光染色显微观察到细胞核在形态上发生了缩缢变形,DNA Ladder检测也出现了标志细胞程序性死亡特征的DNA片断,表明了愈伤组织形成过程中发生了细胞程序性死亡（programmed cell death, PCD）,而在诱导的第六个时期（13 d）DNA的脱尾现象最为明显,表明这一时期是PCD发生较为活跃的时期。利用RT-PCR法在诱导的前九个时期,即诱导的第3、5、7、9、11、13、15、17、19 d的叶片的愈伤组织中均扩增出了细胞程序性死亡相关Beclin1基因的cDNA片段(678 bp),Northen杂交结果显示,在诱导的第13 d,类Beclin1基因表达较强。通过酶联免疫法测定诱导前14 d叶片中内源激素ABA的含量变化,在诱导的第六时期ABA含量相对达到了峰值,结果表明：在烟草叶片愈伤组织诱导过程中伴随PCD的发生,在此过程中内源激素ABA与Beclin1基因起到了调节作用。     

Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

Sites and regulation of polyamine catabolism in the tobacco plant. Correlations with cell division/expansion, cell cycle progression, and vascular development

We previously gave a picture of the homeostatic characteristics of polyamine (PA) biosynthesis and conjugation in tobacco (Nicotiana tabacum) plant organs during development. In this work, we present the sites and regulation of PA catabolism related to cell division/expansion, cell cycle progression, and vascular development in the tobacco plant. Diamine oxidase (DAO), PA oxidase (PAO), peroxidases (POXs), and putrescine N-methyltransferase expressions follow temporally and spatially discrete patterns in shoot apical cells, leaves (apical, peripheral, and central regions), acropetal and basipetal petiole regions, internodes, and young and old roots in developing plants. DAO and PAO produce hydrogen peroxide, a plant signal molecule and substrate for POXs. Gene expression and immunohistochemistry analyses reveal that amine oxidases in developing tobacco tissues precede and overlap with nascent nuclear DNA and also with POXs and lignification. In mature and old tissues, flow cytometry indicates that amine oxidase and POX activities, as well as pao gene and PAO protein levels, coincide with G2 nuclear phase and endoreduplication. In young versus the older roots, amine oxidases and POX expression decrease with parallel inhibition of G2 advance and endoreduplication, whereas putrescine N-methyltransferase dramatically increases. In both hypergeous and hypogeous tissues, DAO and PAO expression occurs in cells destined to undergo lignification, suggesting a different in situ localization. DNA synthesis early in development and the advance in cell cycle/endocycle are temporally and spatially related to PA catabolism and vascular development.